Chronic Neurological Disease Due to Methylmercury Poisoning.

Organic mercury, especially methylmercury, poisoning causes chronic neurological disease predominantly affecting the brain. There have been documented exposures from eating fish from contaminated waters in Japan and in northwestern Ontario and in Iraq from eating bread made from seed wheat treated with methylmercuric fungicide. The neurological disease is called Minamata disease in Japan. Visual field constriction due to involvement of the calcarine cortex, sensory disturbance due to involvement of the somatosensory cortex, and cerebellar ataxia due to involvement of granule cell neurons of the cerebellum are common and characteristic features due to methylmercury poisoning. Other neurological features include dysarthria, postural and action tremor, cognitive impairment, and hearing loss and dysequilibrium. In contrast, peripheral nerve disease is more characteristic of inorganic mercury intoxication. Similarly, psychosis is more typical of exposure to inorganic mercury, which has been documented in the felt hat industry ("mad hatter"). Laboratory tests (e.g., on blood and hair and toenail samples) are of limited value in the assessment of chronic neurological disease due to mercury poisoning because they may not reflect remote neuronal injury due to mercury. Methylmercury also causes injury to fetal brains during development. There is no effective treatment.

Lyssavirus phosphoproteins increase mitochondrial complex I activity and levels of reactive oxygen species.

We have previously demonstrated that serine residues at positions 162 and 166 of the rabies virus (RABV) phosphoprotein (P) are critical for oxidative stress induced by CVS in cultured cells. We have now evaluated the P of two street RABV variants and Mokola (MOK) virus. The P of these viruses, like CVS, induces an increase in complex I activities and reactive oxygen species levels in transfected cells. Although the sequence homology of P is only 45% with MOK (higher for street viruses) and CVS, serine residues are conserved at positions 162 and 166, suggesting their potential importance in oxidative stress.

Serine residues at positions 162 and 166 of the rabies virus phosphoprotein are critical for the induction of oxidative stress in rabies virus infection.

Our previous work in a mouse model of experimental rabies showed neuronal process (dendrites and axons) degeneration in association with severe clinical disease. Cultured adult rodent dorsal root ganglion (DRG) neurons infected with the challenge virus standard-11 (CVS) strain of rabies virus (RABV) showed axonal swellings and reduced axonal growth with evidence of oxidative stress. We have shown that CVS infection alters a variety of mitochondrial parameters and increases mitochondrial complex I activity and reactive oxygen species (ROS) production. Expression of a peptide from amino acid 139-172 of the CVS phosphoprotein (P) increased complex I activity and ROS generation similar to expression of the entire P. Site-directed mutational analyses illustrated the importance of the 145-151 and 157-169 regions of P and that serine residues at 162 and 166 are important single amino acid sites. Two CVS recombinant viruses with serine to alanine mutations at positions 162 (A162r) and 166 (A166r) did not increase complex I activity or ROS generation and also did not induce axonal swellings or inhibit axonal growth in DRG neurons. RABV infection is a mitochondrial disorder initiated by interaction of the RABV P and complex I; S162 and S166 are critical sites in the P for this interaction. The resulting mitochondrial dysfunction produces oxidative stress in neurons causing acute degenerative changes affecting neuronal processes resulting in a severe and fatal clinical disease. This information will be important for the future development of novel therapies for rabies.

Cerebrospinal Fluid Pleocytosis in Critical Care Patients With Seizures.

OBJECTIVES: To assess the etiology of cerebrospinal fluid (CSF) pleocytosis in critical care patients with seizure(s) or status epilepticus (SE). Many previous studies, some performed decades ago, concluded that CSF pleocytosis may be entirely attributable to seizure activity. METHODS: We undertook a retrospective chart review of adult patients with an admitting or acquired diagnosis of seizure(s) or SE in critical care units at the Winnipeg Health Sciences Centre between 2009 and 2012. Patients were identified through a critical care information database at a tertiary care center. We limited our study to patients who had lumbar punctures at our center within 5 days of seizure(s) or SE. RESULTS: Of 426 patients with seizures in critical care units, 51 met the inclusion criteria. Seizure subtypes included focal seizures (5 or 10%), generalized seizures (14 or 27%), and SE (32 or 63%). Twelve (seven with SE) of the 51 (24%) were found to have CSF pleocytosis. A probable etiological cause for the CSF pleocytosis was identified in all 12 cases. CONCLUSIONS: We conclude that seizures do not directly induce a CSF pleocytosis. Instead, the CSF pleocytosis more likely reflects the underlying acute or chronic brain process responsible for the seizure(s). This was not readily apparent in early studies without magnetic resonance imaging (MRI) of the brain and currently available laboratory investigations. An etiological cause of CSF pleocytosis must always be sought when patients present with seizures and it should never be assumed that seizures are the cause.

Diabolical effects of rabies encephalitis.

Rabies is an acute encephalomyelitis in humans and animals caused by rabies virus (RABV) infection. Because the neuropathological changes are very mild in rabies, it has been assumed that neuronal dysfunction likely explains the severe clinical disease. Recently, degenerative changes have been observed in neuronal processes (dendrites and axons) in experimental rabies. In vitro studies have shown evidence of oxidative stress that is caused by mitochondrial dysfunction. Recent work has shown that the RABV phosphoprotein (P) interacts with mitochondrial Complex I leading to overproduction of reactive oxygen species, which results in injury to axons. Amino acids at positions 139 to 172 of the P are critical in this process. Rabies vectors frequently show behavioral changes. Aggressive behavior with biting is important for transmission of the virus to new hosts at a time when virus is secreted in the saliva. Aggression is associated with low serotonergic activity in the brain. Charlton and coworkers performed studies in experimentally infected striped skunks with skunk rabies virus and observed aggressive behavioral responses. Heavy accumulation of RABV antigen was found in the midbrain raphe nuclei, indicating that impaired serotonin neurotransmission from the brainstem may account for the aggressive behavior. We now have an improved understanding of how RABV causes neuronal injury and how the infection results in behavioral changes that promote viral transmission to new hosts.

Critical Appraisal of the Milwaukee Protocol for Rabies: This Failed Approach Should Be Abandoned.

The Milwaukee protocol has been attributed to survival in rabies encephalitis despite a lack of scientific evidence supporting its therapeutic measures. We have reviewed the literature with reference to specific treatment recommendations made within the protocol. Current literature fails to support an important role for excitotoxicity and cerebral vasospasm in rabies encephalitis. Therapies suggested in the Milwaukee protocol include therapeutic coma, ketamine infusion, amantadine, and the screening/prophylaxis/management of cerebral vasospasm. None of these therapies can be substantiated in rabies or other forms of acute viral encephalitis. Serious concerns over the current protocol recommendations are warranted. The recommendations made by the Milwaukee protocol warrant serious reconsideration before any future use of this failed protocol.

Pandemic H1N1 Vaccination and Incidence of Acute Disseminated Encephalomyelitis in Manitoba.

BACKGROUND: An increased incidence of hospital admissions coded as acute disseminated encephalomyelitis (ADEM) was noted in Winnipeg, Manitoba, Canada, during the second wave of the influenza pandemic from October 2009 to March 2010. However, it was not clear whether this was due to heightened awareness of potential neurological complications of influenza or influenza vaccination or an actual increase in the number of cases. METHODS: We extracted data from the charts of 139 patients hospitalized with an International Classification of Diseases-10 discharge code indicating ADEM (G04.0) or unspecified noninfectious encephalitis or myelitis (G04.8, G04.9) between January 2006 and December 2012. Clinical and laboratory data were reviewed by a neurologist, and diagnoses were determined using the Brighton criteria. RESULTS: Over the entire study period, there were 22 cases of ADEM. During the peak pandemic period (April-December 2009), seven patients were hospitalized with ADEM, corresponding to a rate of 7.8/million/year; 4.7 (95% confidence interval: 1.9-11.4) times higher than the rate before or after the pandemic period. Only one patient with ADEM had received the monovalent A(H1N1)pdm09 vaccine within 12 weeks of hospitalization. CONCLUSIONS: We have found an increased incidence of ADEM during the pandemic period that may be related, at least in part, to the increased incidence of influenza during that period. However, there was no temporal relationship with the administration of A(H1N1)pdm09 or seasonal influenza vaccines. Our study provides reassurance that use of these vaccines was not associated with increased risk of ADEM.

Herpes Simplex Encephalitis: Lack of Clinical Benefit of Long-term Valacyclovir Therapy.

BACKGROUND: Despite the proven efficacy of acyclovir (ACV) therapy, herpes simplex encephalitis (HSE) continues to cause substantial morbidity and mortality. Among patients with HSE treated with ACV, the mortality rate is approximately 14%-19%. Among survivors, 45%-60% have neuropsychological sequelae at 1 year. Thus, improving therapeutic approaches to HSE remains a high priority. METHODS: Following completion of a standard course of intravenous ACV, 87 adult patients with HSE (confirmed by positive polymerase chain reaction [PCR] for herpes simplex virus DNA in cerebrospinal fluid) were randomized to receive either valacyclovir (VACV) 2 g thrice daily (n = 40) or placebo tablets (n = 47) for 90 days (12 tablets of study medication daily). The primary endpoint was survival with no or mild neuropsychological impairment at 12 months, as measured by the Mattis Dementia Rating Scale (MDRS). Logistic regression was utilized to assess factors related to the primary endpoint. RESULTS: The demographic characteristics of the 2 randomization groups were statistically similar with no significant differences in age, sex, or race. At 12 months, there was no significant difference in the MDRS scoring for VACV-treated vs placebo recipients, with 85.7% and 90.2%, respectively, of patients demonstrating no or mild neuropsychological impairment (P = .72). No significant study-related adverse events were encountered in either treatment group. CONCLUSIONS: Following standard treatment with intravenous ACV for PCR-confirmed HSE, an additional 3-month course of oral VACV therapy did not provide added benefit as measured by neuropsychological testing 12 months later in a population of relatively high-functioning survivors. CLINICAL TRIALS REGISTRATION: NCT00031486.

Current status of rabies and prospects for elimination.

Rabies is one of the most deadly infectious diseases, with a case-fatality rate approaching 100%. The disease is established on all continents apart from Antarctica; most cases are reported in Africa and Asia, with thousands of deaths recorded annually. However, the estimated annual figure of almost 60,000 human rabies fatalities is probably an underestimate. Almost all cases of human rabies result from bites from infected dogs. Therefore, the most cost-effective approach to elimination of the global burden of human rabies is to control canine rabies rather than expansion of the availability of human prophylaxis. Mass vaccination campaigns with parenteral vaccines, and advances in oral vaccines for wildlife, have allowed the elimination of rabies in terrestrial carnivores in several countries worldwide. The subsequent reduction in cases of human rabies in such regions advocates the multidisciplinary One Health approach to rabies control through the mass vaccination of dogs and control of canine populations.

Rabies virus phosphoprotein interacts with mitochondrial Complex I and induces mitochondrial dysfunction and oxidative stress.

Our previous studies in an experimental model of rabies showed neuronal process degeneration in association with severe clinical disease. Cultured adult rodent dorsal root ganglion neurons infected with challenge virus standard (CVS)-11 strain of rabies virus (RABV) showed axonal swellings and reduced axonal growth with evidence of oxidative stress. We have shown that CVS infection alters a variety of mitochondrial parameters and increases reactive oxygen species (ROS) production and mitochondrial Complex I activity vs. mock infection. We have hypothesized that a RABV protein targets mitochondria and triggers dysfunction. Mitochondrial extracts of mouse neuroblastoma cells were analyzed with a proteomics approach. We have identified peptides belonging to the RABV nucleocapsid protein (N), phosphoprotein (P), and glycoprotein (G), and our data indicate that the extract was most highly enriched with P. P was also detected by immunoblotting in RABV-infected purified mitochondrial extracts and also in Complex I immunoprecipitates from the extracts but not in mock-infected extracts. A plasmid expressing P in cells increased Complex I activity and increased ROS generation, whereas expression of other RABV proteins did not. We have analyzed recombinant plasmids encoding various P gene segments. Expression of a peptide from amino acid 139-172 increased Complex I activity and ROS generation similar to expression of the entire P protein, whereas peptides that did not contain this region did not increase Complex I activity or induce ROS generation. These results indicate that a region of the RABV P interacts with Complex I in mitochondria causing mitochondrial dysfunction, increased generation of ROS, and oxidative stress.

Metagenomic and metabolomic characterization of rabies encephalitis: new insights into the treatment of an ancient disease.

Rabies virus (RV) infection is a fatal nervous system disorder. We describe a patient who died of rabies despite a neuroprotective intervention. Neuropathology showed neuronal loss with abundant RV antigen, genome, and Negri bodies, accompanied by intense neuroinflammation, including by CD8(+) T lymphocyte infiltrates. Deep sequencing and real-time reverse-transcription polymerase chain reaction revealed RNA encoding a bat RV strain together with inflammatory gene induction. RV-infected brain demonstrated reduced neuronal metabolites with an anaerobic metabolic profile by nuclear magnetic resonance (NMR) spectroscopy. These multiplatform studies highlight the extent of ongoing viral replication coupled with inflammation in treated rabies, indicative of a neurological immune reconstitution inflammatory syndrome.

Randomized, double-blind, controlled trial of a combination of alpha-lipoic acid and pregabalin for neuropathic pain: the PAIN-CARE trial.

ABSTRACT: We compared a combination of the nonsedating antioxidant, alpha-lipoic acid (ALA), with the sedating anticonvulsant, pregabalin, vs each monotherapy to treat neuropathic pain due to peripheral neuropathies. In this randomized, double-blind, 3-period crossover trial, participants received oral ALA, pregabalin, and their combination-each for 6 weeks. The primary outcome was mean daily pain intensity at maximal tolerated doses (MTD); secondary outcomes included quality of life (SF-36), sleep (Medical Outcomes Study-Sleep Scale), adverse effects, drug doses, and other measures. Of 55 participants randomized (20-diabetic neuropathy, 19-small fiber neuropathy, and 16-other neuropathies), 46 completed 2 periods, and 44 completed 3. At MTD, the primary outcome of mean pain intensity (0-10) was 5.32 (standard error, SE = 0.18), 3.96 (0.25), 3.25 (0.25), and 3.16 (0.25) at baseline, ALA, pregabalin, and combination, respectively ( P < 0.01 for ALA vs combination and pregabalin). Treatment differences were similar in subgroups with diabetic neuropathy and with other neuropathies. SF-36 total scores (higher number indicates better quality of life) were 66.6 (1.88), 70.1 (1.88), and 69.4 (1.87) with ALA, pregabalin, and combination ( P < 0.05 for ALA vs combination and pregabalin). At MTD, there were no statistically significant treatment differences in adverse effects or drug doses. This trial demonstrates superiority of pregabalin vs ALA but provides no evidence to suggest added benefit of combining ALA with pregabalin to treat neuropathic pain.

Clinical features of dog- and bat-acquired rabies in humans.

BACKGROUND: Clinical differences in rabies due to canine and bat rabies virus variants have been noted, but no detailed studies have been reported to support these observations. METHODS: Using the Morbidity and Mortality Weekly Report and PubMed, we identified 142 case reports of rabies from North America, South America, Europe, Africa, and Asia. We systematically abstracted 126 selected data elements and compared clinical features and investigation results in dog- and bat-acquired cases of rabies. RESULTS: Survivors and cases acquired from aerosolized viral exposure or tissue/organ transplant were excluded (n = 20). Of 122 cases, 49 (40.2%) were dog-acquired and 54 (44.3%) were bat-acquired. Bat-acquired cases of rabies were more often misdiagnosed and lacked a bite history. Encephalopathy, hydrophobia, and aerophobia were more common in dog-acquired rabies. Abnormal cranial nerve, motor, and sensory examinations, tremor, myoclonus, local sensory symptoms, symptoms at the exposure site, and local symptoms in the absence of a bite or scratch were more common in patients with bat-acquired rabies, as was increased cerebrospinal fluid protein (P = .031). Patients with paralytic rabies had longer survival times than those with encephalitic rabies, and also had shorter incubation periods if they had received postexposure prophylaxis. CONCLUSIONS: Clinical differences in dog- and bat-acquired rabies may reflect differences in the route of viral spread of rabies virus variants in the nervous system, although certain variants could cause more severe dysfunction in neuronal subpopulations. Recognition that bat-acquired rabies may present with different clinical manifestations than dog-acquired rabies may help improve the early diagnosis of rabies.

Role of nuclear factor-κB in oxidative stress associated with rabies virus infection of adult rat dorsal root ganglion neurons.

Recent studies in an experimental model of rabies showed major structural changes in the brain involving neuronal processes that are associated with severe clinical disease. Cultured adult rat dorsal root ganglion (DRG) neurons infected with the challenge virus standard-11 strain of rabies virus (CVS) showed axonal swellings and immunostaining for 4-hydroxy-2-nonenal (4-HNE), indicating evidence of lipid peroxidation associated with oxidative stress and reduced axonal growth compared to that of mock-infected DRG neurons. We have evaluated whether nuclear factor (NF)-κB might act as a critical bridge linking CVS infection and oxidative stress. On Western immunoblotting, CVS infection induced expression of the NF-κB p50 subunit compared to that of mock infection. Ciliary neurotrophic factor, a potent activator of NF-κB, had no effect on mock-infected rat DRG neurons and reduced the number of 4-HNE-labeled puncta. SN50, a peptide inhibitor of NF-κB, and CVS infection had an additive effect in producing axonal swellings, indicating that NF-κB is neuroprotective. The fluorescent signal for subunit p50 was quantitatively evaluated in the nucleus and cytoplasm of mock- and CVS-infected rat DRG neurons. At 24 h postinfection (p.i.), there was a significant increase in the nucleus/cytoplasm ratio, indicating increased transcriptional activity of NF-κB, perhaps as a response to stress. At both 48 and 72 h p.i., there was significantly reduced nuclear localization of NF-κB. CVS infection may induce oxidative stress by inhibiting nuclear activation of NF-κB. A rabies virus protein may directly inhibit NF-κB activity. Further investigations are needed to gain a better understanding of the basic mechanisms involved in the oxidative damage associated with rabies virus infection.

Mitochondrial dysfunction in rabies virus infection of neurons.

Infection with the challenge virus standard-11 (CVS) strain of fixed rabies virus induces neuronal process degeneration in adult mice after hindlimb footpad inoculation. CVS-induced axonal swellings of primary rodent dorsal root ganglion neurons are associated with 4-hydroxy-2-nonenal protein adduct staining, indicating a critical role of oxidative stress. Mitochondrial dysfunction is the major cause of oxidative stress. We hypothesized that CVS infection induces mitochondrial dysfunction leading to oxidative stress. We investigated the effects of CVS infection on several mitochondrial parameters in different cell types. CVS infection significantly increased maximal uncoupled respiration and complex IV respiration and complex I and complex IV activities, but did not affect complex II-III or citrate synthase activities. Increases in complex I activity, but not complex IV activity, correlated with susceptibility of the cells to CVS infection. CVS infection maintained coupled respiration and rate of proton leak, indicating a tight mitochondrial coupling. Possibly as a result of enhanced complex activity and efficient coupling, a high mitochondrial membrane potential was generated. CVS infection reduced the intracellular ATP level and altered the cellular redox state as indicated by a high NADH/NAD+ ratio. The basal production of reactive oxygen species (ROS) was not affected in CVS-infected neurons. However, a higher rate of ROS generation occurred in CVS-infected neurons in the presence of mitochondrial substrates and inhibitors. We conclude that CVS infection induces mitochondrial dysfunction leading to ROS overgeneration and oxidative stress.

Improvement in thrombolytic therapy administration in acute stroke with feedback.

BACKGROUND: The benefits of intravenous recombinant tissue plasminogen activator (rt-PA) in acute ischemic stroke is time dependent. Guidelines recommend a door-to-needle (DTN) time of less than 60 minutes. METHODS: A retrospective audit of 730 stroke charts from 2008 - 2011 was conducted at Health Sciences Centre. 158 patients treated with IV rt-PA were identified. The time intervals between Emergency Department (ED) arrival, administration of rt-PA and uninfused brain computed axial tomographic scan (CT) were recorded. From this, CT to needle times were calculated. During November 2010 to January 2011 feedback was given to neurologists, ED physicians, ED nurses, and CT technologists. This raised awareness and emphasized the importance of this time driven protocol. RESULTS: The median DTN times for 2008, 2009, and 2010 were 69, 71 and 76 minutes respectively. The median CT-to-needle time for this time period was 47 minutes. In 2011 (n =58) the median DTN time was 49 minutes and the median CT-to-needle was 18 minutes, which were marked improvements (p<0.00005 and p<0.005, respectively). In 2008-2010 only 31% of treated patients (n=100) received rt-PA within 60 minutes, whereas in 2011 this increased to 64%. CONCLUSIONS: Dramatic improvements in DTN times and in the percentage of patients receiving rt-PA treatment within 60 minutes were observed in 2011 after feedback was provided regarding the suboptimal performance. Prior to receiving feedback, DTN times were similar to national median DTN times. All centres administering rt-PA for acute ischemic stroke should monitor their clinical performance and give feedback on a regular basis.

Delays in initiation of acyclovir therapy in herpes simplex encephalitis.

BACKGROUND: Diagnosis of herpes simplex encephalitis (HSE) is based on clinical findings, MRI, and detection of herpes simplex virus (HSV) DNA in cerebrospinal fluid (CSF) using polymerase chain reaction amplification. Delays in starting treatment are associated with poorer clinical outcomes. We assessed the timing of initiation of acyclovir therapy in HSE. METHODS: Inpatient databases from seven hospitals in Winnipeg, Manitoba were used to identify individuals diagnosed with encephalitis and HSE from 2004 to 2009. The time taken to initiate therapy with acyclovir and the reasons for delays were determined. RESULTS: Seventy-seven patients were identified; 69 (90%) received acyclovir; in the others a non-HSV infection was strongly suspected. Thirteen patients were subsequently confirmed to have HSE. Acyclovir was initiated a median of 21 hours (3-407) after presentation in encephalitis cases, and a median of 11 hours (3-118) in HSE. The most common reason for delay was a failure to consider HSE in the differential diagnosis, despite suggestive clinical features. Where therapy was delayed in HSE patients, the decision to begin acyclovir was prompted by transfer of the patient to a different service (55%), recommendations by consultants (18%), imaging results (18%), and CSF pleocytosis (9%). CONCLUSIONS: Delays in initiating acyclovir for HSE are common, and are most often due to a failure to consider HSE in a timely fashion on presentation. In order to improve patient outcomes, physicians should be more vigilant for HSE, and begin acyclovir therapy expeditiously on the basis of clinical suspicion rather than waiting for confirmatory tests.