Preparation of human formalin-fixed brain slices for electron microscopic investigations.

Ultra-structural analysis of human post-mortem brain tissue is important for investigations into the pathomechanism of neuropsychiatric disorders, especially those lacking alternative models of studying human-specific morphological features. For example, Von Economo Neurons (VENs) mainly located in the anterior cingulate cortex and in the anterior part of the insula, which seem to play a role in a variety of neuropsychiatric conditions, including frontotemporal dementia, autism and schizophrenia, can hardly be studied in nonhuman animals. Accordingly, little is known about the ultra-structural alterations of these neurons, though important research using qualitative stereological methods has revealed that protein expression of the VENs assigns them a role in immune function. Formaldehyde, which is the most common fixative in human pathology, interferes with the immunoreactivity of the tissue, possibly leading to unreliable results. Therefore, a method for ultra-structural investigations independent of antigenic properties of the fixated tissue is needed. Here, we propose an approach using electron microscopy to examine cytoskeletal structures, synapses and mitochondria in these cells. We also show that our methodology is able to keep tissue consumption to a minimum, while still allowing for the specimens to be handled with ease by using agar embedded slices in contrast to blocks for the embedding procedure. Accordingly, a stepwise protocol utilising 60µm thick human post mortem brain sections for electron microscopic ultra-structural investigations is presented.

Influenza caused epidemic encephalitis (encephalitis lethargica): the circumstantial evidence and a challenge to the nonbelievers.

Twenty years ago, circumstantial evidence was compiled to link the 1918 pandemic influenza virus to a CNS disorder called epidemic encephalitis or encephalitis lethargica. A challenge was issued to naysayers. During the past two decades, the knowledge about the influenza virus and the 1918 pandemic virus in particular has had dramatic advancement. The 1918 virus has been resurrected and reconstructed. Experimental studies of mice inoculated with a neurovirulent avian virus have delineated the neuropathology of influenza encephalitis. Review of autopsy cases of encephalitis lethargica revealed that the neuropathology during and shortly after the pandemic was unique. Surprisingly two different viruses were involved with the great pandemic. A single amino acid difference in the hemagglutinin of the two viruses changed the preferred receptor of the virus in the host cell. One virus has qualities that suggest that it is neurovirulent. Circumstantial evidence suggests that the cause of death in some influenza patients was neurogenic congestive heart failure with pulmonary edema. Theories about the pathophysiology of encephalitis lethargica and postencephalitic Parkinson's disease are offered.

Review: neuropathology of acute phase encephalitis lethargica: a review of cases from the epidemic period.

INTRODUCTION: Encephalitis lethargica (EL), an epidemic disease of the early 20th century, has continued to be diagnosed sporadically since that time, including a report of 20 new cases in 2004. Many of the recent case reports state that the primary neuropathology of acute EL consists of inflammatory changes and lesions within the midbrain, basal ganglia and substantia nigra. However, the neuropathology of acute EL cases from the epidemic period was actually much more widespread. METHODS: In order to characterize the neuropathology of acute phase EL, we developed a database of EL pathology based on 112 cases from the years 1915 to 1940, of which most died within 2 weeks of EL onset. RESULTS: Our analysis revealed that cortical damage was prevalent in 75% of the 112 cases; damage to the meninges and brainstem occurred in approximately half of the cases; and the substantia nigra was damaged in only 13% of these acute cases. We also found that after 1921, damage to cranial nerve nuclei was not reported. An analysis of the neuropathology and clinical symptoms revealed little correlation. CONCLUSIONS: Based on these findings, putative modern cases of acute EL with MRI/CT indicated lesions confined solely to the midbrain, brainstem, and/or basal ganglia should not be considered, consistent with that reported during epidemic period.

Absence of alpha-synuclein pathology in postencephalitic parkinsonism.

Postencephalitic parkinsonism (PEP), a chronic complication of encephalitis lethargica, is a tauopathy characterized by multisystem neuronal loss and gliosis with widespread neurofibrillary lesions composed of both 3- and 4-repeat (3R and 4R) tau isoforms. Previous immunohistochemical studies in a small number of PEP cases demonstrated absence of Lewy bodies as well as the lack of other alpha-synuclein pathology, classifying PEP as a "pure" tauopathy. Neuropathologic examination of 10 brains with clinico-pathologically verified PEP confirmed widespread neurodegeneration in subcortical and brainstem areas associated with multifocal neurofibrillary pathology comprising both 3R and 4R tau. Very rare beta-amyloid deposits were observed in two elderly patients, while Lewy bodies and neurites or any other alpha-synuclein deposits were completely absent. The causes and molecular background of total absence of alpha-synuclein pathology in PEP, in contrast to most other tauopathies, remain as unknown as the pathogenesis of PEP.

From juvenile parkinsonism to encephalitis lethargica, a new phenotype of post-streptococcal disorders: case report.

We report the case of a 16-year-old boy presented with a mild akinetic-rigid parkinsonism shortly after a post-streptococcal infection. After stopping corticoids, he had a rapid neurological deterioration to a fatal encephalitis lethargica-like syndrome. Serum analysis demonstrated consistently elevated anti-streptolysin-O. This case illustrates a new severe phenotype in the spectrum of the post-streptococcal disorders. This etiology should be considered in the differential diagnosis of a movement disorder with a rapid detrimental evolution.

Constantin von Economo's contribution to the understanding of movement disorders.

Constantin von Economo's (CvE) main scientific achievements were his studies on the cytoarchitectonics of the cerebral cortex, sleep, and encephalitis lethargica (EL). He found a close relationship between motor symptoms and psychiatric and behavioral disorders in EL and postencephalitic Parkinsonism and identified the underlying neuropathology in the diencephalon and the brainstem. In agreement with Tretiakoff's findings in Parkinson's disease, CvE related postencephalitic Parkinsonism to neuronal loss in the substantia nigra. Several of CvE's early, less well-known publications also deal with the basal ganglia and movement disorders. He demonstrated in rabbits that the substantia nigra modulates automatization, coordination, and succession of masticatory movements and swallowing. In a study on the effects of experimental lesions of the cerebral peduncle in cats and monkeys, CvE hypothesized a corticotegmental pathway that maintains motor functions after pyramidal tract lesions. Recent studies have identified this pathway, which ends in the pedunculopontine nucleus. In a study on posthemiplegic chorea, CvE discussed various pathophysiological hypotheses that partly resemble modern concepts of chorea. In a clinicopathological study on Wilson's disease, CvE traced the striofugal fibers and visualized the basal ganglia outflow pathways. CvE was an outstanding multidisciplinary movement disorder specialist who contributed substantially to modern basal ganglia research.

Encephalitis lethargica syndrome: 20 new cases and evidence of basal ganglia autoimmunity.

In 1916, von Economo first described encephalitis lethargica (EL), a CNS disorder presenting with pharyngitis followed by sleep disorder, basal ganglia signs (particularly parkinsonism) and neuropsychiatric sequelae. Since the 1916-1927 epidemic, only sporadic cases have been described. Pathological studies revealed an encephalitis of the midbrain and basal ganglia, with lymphocyte (predominantly plasma cell) infiltration. The EL epidemic occurred during the same time period as the 1918 influenza pandemic, and the two outbreaks have been linked in the medical literature. However, von Economo and other contemporary scientists thought that the 1918 influenza virus was not the cause of EL. Recent examination of archived EL brain material has failed to demonstrate influenza RNA, adding to the evidence that EL was not an invasive influenza encephalitis. By contrast, the findings of intrathecal oligoclonal bands (OCB) and beneficial effects of steroid treatments have provoked the hypothesis that EL may be immune-mediated. We have recently seen 20 patients with a similar EL phenotype, 55% of whom had a preceding pharyngitis. The patients had remarkable similarity to the historical descriptions of EL: sleep disorder (somnolence, sleep inversion or insomnia), lethargy, parkinsonism, dyskinesias and neuropsychiatric symptoms. CSF examination commonly showed elevated protein and OCB (75 and 69% respectively). Investigation found no evidence of viral encephalitis or other recognized causes of rapid-onset parkinsonism. MRI of the brain was normal in 60% but showed inflammatory changes localized to the deep grey matter in 40% of patients. We investigated the possibility that this phenotype could be a postinfectious autoimmune CNS disorder, and therefore similar to Sydenham's chorea. Anti-streptolysin-O titres were elevated in 65% of patients. Furthermore, western immunoblotting showed that 95% of EL patients had autoantibodies reactive against human basal ganglia antigens. These antibodies were also present in the CSF in four patients tested. By contrast, antibodies reactive against the basal ganglia were found in only 2-4% of child and adult controls (n = 173, P < 0.0001). Rather than showing polyspecific binding, these antibodies bound to common neural autoantigens of molecular weight 40, 45, 60 and 98 kDa. Regional tissue comparisons showed that the majority of these autoantigens were specific to or enriched in CNS tissue. Immunohistochemistry with secondary staining localized antibody binding to neurons rather than glial populations. Further investigation is required to determine whether these antibodies affect neuronal function (i.e. whether they are pathogenic anti-neuronal antibodies). Histopathology in one case demonstrated striatal encephalitis with perivenous B- and T-lymphocytic infiltration. We believe an EL-like syndrome is still prevalent, and propose that this syndrome may be secondary to autoimmunity against deep grey matter neurons.

Lack of detection of influenza genes in archived formalin-fixed, paraffin wax-embedded brain samples of encephalitis lethargica patients from 1916 to 1920.

A method was developed for detection of influenza genes in formalin-fixed brains of mice that had been experimentally infected with influenza A/NWS/33 (H1N1) virus. Using this technique, messenger ribonucleic acid (mRNA) of the beta-actin gene was detected in eight clinical brain samples from the 1916-1920 outbreak of encephalitis lethargica, showing preservation of particular mRNAs. However, we did not detect influenza nucleotide sequences of M, NP, and NS genes from these same samples. We conclude either that influenza was not the causative agent of encephalitis lethargica or, possibly, that the virus had a hit-and-run mechanism and was no longer present in the brain at the time of death of the patients.

Influenza RNA not detected in archival brain tissues from acute encephalitis lethargica cases or in postencephalitic Parkinson cases.

Encephalitis lethargica (EL) was a mysterious epidemic. temporally associated with the 1918 Spanish influenza pandemic. Numerous symptoms characterized this disease, including headache, diplopia, fever, fatal coma, delirium, oculogyric crisis, lethargy, catatonia, and psychiatric symptoms. Many patients who initially recovered subsequently developed profound, chronic parkinsonism. The etiologic association of influenza with EL is controversial. Five acute EL autopsies and more than 70 postencephalitic parkinsonian autopsies were available in the Armed Forces Institute of Pathology (AFIP) tissue repository. Two of these 5 acute EL cases had histopathologic changes consistent with that diagnosis. The remaining 3 cases were classified as possible acute EL cases as the autopsy material was insufficient for detailed histopathologic examination. RNA lysates were prepared from 29 CNS autopsy tissue blocks from the 5 acute cases and 9 lysates from blocks containing substantia nigra from 2 postencephalitic cases. RNA recovery was assessed by amplification of beta-2-microglobulin mRNA and 65% of the tissue blocks contained amplifiable RNA. Reverse transcription-polymerase chain reaction (RT-PCR) for influenza matrix and nucleoprotein genes was negative in all cases. Thus, it is unlikely that the 1918 influenza virus was neurotropic and directly responsible for the outbreak of EL.