Pseudorabies virus encephalitis in humans: a case series study.

Pseudorabies virus (PRV) is known to cause severe encephalitis in juvenile pigs and various non-native hosts; recent evidences suggest that PRV might cause encephalitis in humans. In a multicenter cohort study in China, next-generation sequencing of cerebrospinal fluid (CSF) was performed to detect pathogens in all patients with clinically suspected central nervous system infections. This study involved all the patients whose CSF samples were positive for PRV-DNA; their clinical features were evaluated, and species-specific PCR and serological tests were sequentially applied for validation. Among the 472 patients tested from June 1, 2016, to December 1, 2018, six were positive for PRV-DNA, which were partially validated by PCR and serological tests. Additionally, we retrospectively examined another case with similar clinical and neuroimaging appearance and detected the presence of PRV-DNA. These patients had similar clinical manifestations, including a rapid progression of panencephalitis, and similar neuroimaging features of symmetric lesions in the basal ganglia and bilateral hemispheres. Six of the patients were engaged in occupations connected with swine production. PRV infection should be suspected in patients with rapidly progressive panencephalitis and characteristic neuroimaging features, especially with exposure to swine.

The regional pattern of abnormal cerebrovascular reactivity in HIV-infected, virally suppressed women.

The purpose of this study was to assess whole brain and regional patterns of cerebrovascular reactivity (CVR) abnormalities in HIV-infected women using quantitative whole brain arterial spin labeling (ASL). We hypothesized that HIV-infected women would demonstrate decreased regional brain CVR despite viral suppression. This cross-sectional study recruited subjects from the Bay Area Women's Interagency Health Study (WIHS)-a cohort study designed to investigate the progression of HIV disease in women. In addition to conventional noncontrast cerebral MRI sequences, perfusion imaging was performed before and after the administration of intravenous acetazolamide. CVR was measured by comparing quantitative ASL brain perfusion before and after administration of intravenous acetazolamide. In order to validate and corroborate ASL-based whole brain and regional perfusion, phase-contrast (PC) imaging was also performed through the major neck vessels. FLAIR and susceptibility weighted sequences were performed to assess for white matter injury and microbleeds, respectively. Ten HIV-infected women and seven uninfected, age-matched controls were evaluated. Significant group differences were present in whole brain and regional CVR between HIV-infected and uninfected women. These regional differences were significant in the frontal lobe and basal ganglia. CVR measurements were not significantly impacted by the degree of white matter signal abnormality or presence of microbleeds. Despite complete viral suppression, dysfunction of the neurovascular unit persists in the HIV population. Given the lack of association between CVR and traditional imaging markers of small vessel disease, CVR quantification may provide an early biomarker of pre-morbid vascular disease.

White matter damage, neuroinflammation, and neuronal integrity in HAND.

HIV-associated neurocognitive disorders (HANDs) persist even with virologic suppression on combination antiretroviral therapy (cART), and the underlying pathophysiological mechanisms are not well understood. We performed structural magnetic resonance imaging and MR spectroscopy (MRS) in HIV+ individuals without major neurocognitive comorbidities. Study participants were classified as neurocognitively unimpaired (NU), asymptomatic (ANI), mild neurocognitive disorder (MND), or HIV-associated dementia (HAD). Using structural MRI, we measured volumes of cortical and subcortical gray matter and total and abnormal white matter (aWM). Using single-voxel MRS, we estimated metabolites in frontal gray matter (FGM) and frontal white matter (FWM) and basal ganglia (BG) regions. Adjusted odds ratios were used to compare HAND to NU. Among 253 participants, 40% met HAND criteria (21% ANI, 15% MND, and 4% HAD). Higher risk of HAND was associated with more aWM. Both HAD and MND also had smaller gray and white matter volumes than NU. Among individuals with undetectable plasma HIV RNA, structural volumetric findings were similar to the overall sample. MND had lower FWM creatine and higher FGM choline relative to NU, whereas HAD and ANI had lower BG N-acetyl aspartate relative to NU. In the virologically suppressed subgroup, however, ANI and MND had higher FGM choline compared to NU. Overall, HAND showed specific alterations (more aWM and inflammation; less gray matter volume and lower NAA). Some MR measures differentiated less severe subtypes of HAND from HAD. These MR alterations may represent legacy effects or accumulating changes, possibly related to medical comorbidities, antiretroviral therapy, or chronic effects of HIV brain infection.

Persistence of SIV in the brain of SIV-infected Chinese rhesus macaques with or without antiretroviral therapy.

Persistence of HIV-1 reservoirs in the central nervous system (CNS) is an obstacle to cure strategies. However, little is known about residual viral distribution, viral replication levels, and genetic diversity in different brain regions of HIV-infected individuals on combination antiretroviral therapy (cART). Because myeloid cells particularly microglia are likely major reservoirs in the brain, and more microglia exist in white matter than gray matter in a human brain, we hypothesized the major viral reservoirs in the brain are the white matter reflected by higher levels of viral DNA. To address the issue, we used the Chinese rhesus macaque (ChRM) model of SIV infection, and treated 11 SIVmac251-infected animals including long-term nonprogressors with cART for up to 24 weeks. SIV reservoirs were assessed by SIV DNA levels in 16 specific regions of the brain and 4 regions of spinal cord. We found relatively high frequencies of SIV in basal ganglia and brain stem compared to other regions. cART-receiving animals had significantly lower SIV DNA levels in the gray matter than white matter. Moreover, a shortened envelope gp120 with 21 nucleotide deletions and guanine-to-adenine hypermutations were observed. These results demonstrate that SIV enters the CNS in SIV-infected ChRM with a major reservoir in the white matter after cART; the SIV/ChRM/cART is an appropriate model for studying HIV CNS reservoirs and testing new eradication strategies. Further, examining multiple regions of the CNS may be needed when assessing whether an agent is successful in reducing the size of SIV reservoirs in the CNS.

Expression of CHRFAM7A and CHRNA7 in neuronal cells and postmortem brain of HIV-infected patients: considerations for HIV-associated neurocognitive disorder.

Despite the recent advances in antiretroviral therapy, human immunodeficiency virus type 1 (HIV-1) remains a global health threat. HIV-1 affects the central nervous system by releasing viral proteins that trigger neuronal death and neuroinflammation, and promotes alterations known as HIV-associated neurocognitive disorders (HAND). This disorder is not fully understood, and no specific treatments are available. Recently, we demonstrated that the HIV-1 envelope protein gp120IIIB induces a functional upregulation of the α7-nicotinic acetylcholine receptor (α7) in neuronal cells. Furthermore, this upregulation promotes cell death that can be abrogated with receptor antagonists, suggesting that α7 may play an important role in the development of HAND. The partial duplication of the gene coding for the α7, known as CHRFAM7A, negatively regulates α7 expression but its role in HIV infection has not been studied. Hence, we studied both CHRNA7 and CHRFAM7A regulation patterns in various gp120IIIB in vitro conditions. In addition, we measured CHRNA7 and CHRFAM7A expression levels in postmortem brain samples from patients suffering from different stages of HAND. Our results demonstrate the induction of CHRNA7 expression accompanied by a significant downregulation of CHRFAM7A in neuronal cells when exposed to pathophysiological concentrations of gp120IIIB. Our results suggest a dysregulation of CHRFAM7A and CHRNA7 expressions in the basal ganglia from postmortem brain samples of HIV+ subjects and expand the current knowledge about the consequences of HIV infection in the brain.

The neuroinvasive profiles of H129 (herpes simplex virus type 1) recombinants with putative anterograde-only transneuronal spread properties.

The use of viruses as transneuronal tracers has become an increasingly powerful technique for defining the synaptic organization of neural networks. Although a number of recombinant alpha herpesviruses are known to spread selectively in the retrograde direction through neural circuits only one strain, the H129 strain of herpes simplex virus type 1, is reported to selectively spread in the anterograde direction. However, it is unclear from the literature whether there is an absolute block or an attenuation of retrograde spread of H129. Here, we demonstrate efficient anterograde spread, and temporally delayed retrograde spread, of H129 and three novel recombinants. In vitro studies revealed no differences in anterograde and retrograde spread of parental H129 and its recombinants through superior cervical ganglion neurons. In vivo injections of rat striatum revealed a clear bias of anterograde spread, although evidence of deficient retrograde transport was also present. Evidence of temporally delayed retrograde transneuronal spread of H129 in the retina was observed following injection of the lateral geniculate nucleus. The data also demonstrated that three novel recombinants efficiently express unique fluorescent reporters and have the capacity to infect the same neurons in dual infection paradigms. From these experiments we conclude that H129 and its recombinants not only efficiently infect neurons through anterograde transneuronal passage, but also are capable of temporally delayed retrograde transneuronal spread. In addition, the capacity to produce dual infection of projection targets following anterograde transneuronal passage provides an important addition to viral transneuronal tracing technology.

Predictors of CNS injury as measured by proton magnetic resonance spectroscopy in the setting of chronic HIV infection and CART.

The reasons for persistent brain dysfunction in chronically HIV-infected persons on stable combined antiretroviral therapies (CART) remain unclear. Host and viral factors along with their interactions were examined in 260 HIV-infected subjects who underwent magnetic resonance spectroscopy (MRS). Metabolite concentrations (NAA/Cr, Cho/Cr, MI/Cr, and Glx/Cr) were measured in the basal ganglia, the frontal white matter, and gray matter, and the best predictive models were selected using a bootstrap-enhanced Akaike information criterion (AIC). Depending on the metabolite and brain region, age, race, HIV RNA concentration, ADC stage, duration of HIV infection, nadir CD4, and/or their interactions were predictive of metabolite concentrations, particularly the basal ganglia NAA/Cr and the mid-frontal NAA/Cr and Glx/Cr, whereas current CD4 and the CPE index rarely or did not predict these changes. These results show for the first time that host and viral factors related to both current and past HIV status contribute to persisting cerebral metabolite abnormalities and provide a framework for further understanding neurological injury in the setting of chronic and stable disease.

Effects of HIV-1 tat on secretion of TNF-α and IL-1ß by U87 cells in AIDS patients with or without AIDS dementia complex.

OBJECTIVE: To explore the role of HIV-1 tat gene variations in AIDS dementia complex (ADC) pathogenesis. METHODS: HIV-1 tat genes derived from peripheral spleen and central basal ganglia of an AIDS patient with ADC and an AIDS patient without ADC were cloned for sequence analysis. HIV-1 tat gene sequence alignment was performed by using CLUSTAL W and the phylogentic analysis was conducted by using Neighbor-joining with MEGA4 software. All tat genes were used to construct recombinant retroviral expressing vector MSCV-IRES-GFP/tat. The MSCV-IRES-GFP/tat was cotransfected into 293T cells with pCMV-VSV-G and pUMVC vectors to assemble the recombinant retrovirus. After infection of gliomas U87 cells with equal amount of the recombinant retrovirus, TNF-α, and IL-1ß concentrations in the supernatant of U87 cells were determined with ELISA. RESULTS: HIV-1 tat genes derived from peripheral spleen and central basal ganglia of the AIDS patient with ADC and the other one without ADC exhibited genetic variations. Tat variations and amino acid mutation sites existed mainly at Tat protein core functional area (38-47aa). All Tat proteins could induce U87 cells to produce TNF-α and IL-1ß, but the level of IL-1ß production was different among Tat proteins derived from the ADC patient's spleen, basal ganglia, and the non-ADC patient's spleen. The level of Tat proteins derived from the ADC patient's spleen, basal ganglia, and the non-ADC patient's spleen were obviously higher than that from the non-ADC patient's basal ganglia. CONCLUSION: Tat protein core functional area (38-47aa) may serve as the key area of enhancing the secretion of IL-1ß. This may be related with the neurotoxicity of HIV-1 Tat.

Progressive cerebral injury in the setting of chronic HIV infection and antiretroviral therapy.

Emerging evidence suggests that CNS injury and neurocognitive impairment persist in the setting of chronic HIV infection and combination antiretroviral therapy (CART). Yet, whether neurological injury can progress in this setting remains uncertain. Magnetic resonance spectroscopy and neurocognitive and clinical assessments were performed over 2 years in 226 HIV-infected individuals on stable CART, including 138 individuals who were neurocognitively asymptomatic (NA). Concentrations of N-acetylaspartate (NAA), creatine (Cr), choline (Cho), myoinositol, and glutamate/glutamine (Glx) were measured in the midfrontal cortex (MFC), frontal white matter (FWM), and basal ganglia (BG). Longitudinal changes in metabolite levels were determined using linear mixed effect models, as were metabolite changes in relation to global neurocognitive function. HIV-infected subjects showed significant annual decreases in brain metabolite levels in all regions examined, including NAA (2.95 %) and Cho (2.61 %) in the FWM; NAA (1.89 %), Cr (1.84 %), Cho (2.19 %), and Glx (6.05 %) in the MFC; and Glx (2.80 %) in the BG. Similar metabolite decreases were observed in the NA and subclinically impaired subgroups, including subjects with virologic suppression in plasma and CSF. Neurocognitive decline was associated with longitudinal decreases in Glx in the FWM and the BG, and in NAA in the BG. Widespread progressive changes in the brain, including neuronal injury, occur in chronically HIV-infected persons despite stable antiretroviral treatment and virologic suppression and can lead to neurocognitive declines. The basis for these findings is poorly understood and warrants further study.

Dysregulation of macrophage-secreted cathepsin B contributes to HIV-1-linked neuronal apoptosis.

Chronic HIV infection leads to the development of cognitive impairments, designated as HIV-associated neurocognitive disorders (HAND). The secretion of soluble neurotoxic factors by HIV-infected macrophages plays a central role in the neuronal dysfunction and cell death associated with HAND. One potentially neurotoxic protein secreted by HIV-1 infected macrophages is cathepsin B. To explore the potential role of cathepsin B in neuronal cell death after HIV infection, we cultured HIV-1(ADA) infected human monocyte-derived macrophages (MDM) and assayed them for expression and activity of cathepsin B and its inhibitors, cystatins B and C. The neurotoxic activity of the secreted cathepsin B was determined by incubating cells from the neuronal cell line SK-N-SH with MDM conditioned media (MCM) from HIV-1 infected cultures. We found that HIV-1 infected MDM secreted significantly higher levels of cathepsin B than did uninfected cells. Moreover, the activity of secreted cathepsin B was significantly increased in HIV-infected MDM at the peak of viral production. Incubation of neuronal cells with supernatants from HIV-infected MDM resulted in a significant increase in the numbers of apoptotic neurons, and this increase was reversed by the addition of either the cathepsin B inhibitor CA-074 or a monoclonal antibody to cathepsin B. In situ proximity ligation assays indicated that the increased neurotoxic activity of the cathepsin B secreted by HIV-infected MDM resulted from decreased interactions between the enzyme and its inhibitors, cystatins B and C. Furthermore, preliminary in vivo studies of human post-mortem brain tissue suggested an upregulation of cathepsin B immunoreactivity in the hippocampus and basal ganglia in individuals with HAND. Our results demonstrate that HIV-1 infection upregulates cathepsin B in macrophages, increases cathepsin B activity, and reduces cystatin-cathepsin interactions, contributing to neuronal apoptosis. These findings provide new evidence for the role of cathepsin B in neuronal cell death induced by HIV-infected macrophages.

Effects of anti-viral therapy and HCV clearance on cerebral metabolism and cognition.

BACKGROUND & AIMS: Chronic hepatitis C virus (HCV) infection is associated with altered cerebral metabolism and cognitive dysfunction. We aimed to evaluate the effect of pegylated interferon/ribavirin (PIFN/R) and HCV clearance on cerebral metabolism, and neuropsychological performance. METHODS: Fifteen non-cirrhotic HCV positive subjects underwent (1)H MR spectroscopy (MRS) before, during, and after treatment with PIFN/R. The metabolites of interest namely, N-acetylaspartate (NAA), choline (Cho), myo-inositol (MI), and the control metabolite creatine (Cr), were acquired from 3 different brain regions; left basal ganglia, left frontal cortex, and left dorso-lateral pre-frontal cortex. Coinciding with this, subjects also underwent a battery of neuropsychological tests to evaluate the domains of verbal learning, memory, attention, language, executive functioning, and motor skills. Seven HCV positive controls (not receiving anti-viral therapy) underwent MRS and neuropsychological testing at two time points, 12 weeks apart, to examine for variation in cerebral metabolites over time and the practice effect of repeat neuropsychological testing. RESULTS: Significant reductions in basal ganglia Cho/Cr (p=0.03) and basal ganglia MI/Cr (p=0.03) were observed in sustained virological responders (SVRs, n=8), but not non-responders/relapsers (NR/R, n=6), indicative of reduced cerebral infection and/or immune activation in those who cleared virus. SVRs demonstrated significant improvements in verbal learning, memory, and visuo-spatial memory. A small but significant improvement in neurocognitive function secondary to the practice effect was seen in both HCV controls and HCV subjects during treatment. CONCLUSIONS: HCV eradication has a beneficial effect on cerebral metabolism and selective aspects of neurocognitive function and is an important factor when contemplating anti-viral therapy in HCV, especially in those with mild disease.

Severe human herpesvirus 6-associated encephalopathy in three children: analysis of cytokine profiles and the carnitine palmitoyltransferase 2 gene.

Three children developed severe encephalopathy associated with human herpesvirus 6 infection. Magnetic resonance imaging of the brain showed either basal ganglia involvement or diffusion abnormalities in the cerebral white matter. Coagulopathy with hypercytokinemia was observed in 2 patients. One demonstrated thermolabile variation in carnitine palmitoyltransferase 2. These results suggest a heterogeneous pathogenic mechanism in encephalopathy associated with human herpesvirus 6 infection.

Alterations in brain metabolism during the first year of HIV infection.

Migration of both uninfected and infected monocytes into the brain during acute HIV infection likely initiates metabolic changes that can be observed with magnetic resonance spectroscopy (MRS). Herein, we measured changes in brain metabolism during the first year of HIV infection and examined the relationship of these metabolite levels to CD16+ monocyte populations measured in the blood. MRS was performed on nine HIV+ subjects identified during acute HIV infection and nine seronegative control subjects. HIV+ subjects were examined within 90 days of an indeterminate Western blot, then again 2 and 6 months later, during early infection. Blood samples were collected for plasma viral RNA and monocyte subset quantification. HIV+ subjects were identified with acute viral ailment and did not display severe cognitive deficits such as dementia or minor cognitive motor disorder. Changes in lipid membrane metabolism (choline levels) in the frontal cortex and white matter were observed during the initial year of HIV infection. Greater numbers of CD16+ monocytes were associated with lower N-acetylaspartate levels and higher choline levels in the brain. These results suggest that HIV infection induces metabolic changes in the brain early during infection and that these changes may be related to monocyte dynamics in the periphery.

Cerebral metabolite abnormalities in human immunodeficiency virus are associated with cortical and subcortical volumes.

Cerebral metabolite disturbances occur among human immunodeficiency virus (HIV)-infected people, and are thought to reflect neuropathology, including proinflammatory processes, and neuronal loss. HIV-associated cortical atrophy continues to occur, though its basis is not well understood, and the relationship of cerebral metabolic disturbance to structural brain abnormalities in HIV has not been well delineated. We hypothesized that metabolite disturbances would be associated with reduced cortical and subcortical volumes. Cerebral volumes were measured in 67 HIV-infected people, including 10 people with mild dementia (acquired immunodeficiency syndrome [AIDS] dimentia complex [ADC] stage >1) via automated magnetic resonance imaging (MRI) segmentation. Magnetic resonance spectroscopy (MRS) was used to measure levels of cerebral metabolites N-acetylaspartate (NAA), myo-inositol (MI), choline-containing compounds (Cho), glutamate/glutamine (Glx), and creatine (Cr) from three brain regions (frontal gray matter, frontal white matter, basal ganglia). Analyses were conducted to examine the associations between MRS and cerebral volumetric measures using both absolute and relative metabolite concentrations. NAA in the mid-frontal gray matter was most consistently associated with cortical (global, frontal, and parietal), ventricular, and caudate volumes based on analysis of absolute metabolite levels, whereas temporal lobe volume was associated with basal ganglia NAA and Glx, and Cho concentrations in the frontal cortex and basal ganglia. Hippocampal volume was associated with frontal white matter NAA, whereas thalamic volume was associated with both frontal white matter NAA and basal ganglia Glx. Analyses of relative metabolite concentrations (referenced to Cr) yielded weaker effects, although more metabolites were retained as significant predictors in the models than the analysis of absolute concentrations. These findings demonstrate that reduced cortical and subcortical volumes, which have been previously found to be linked to HIV status and history, are also strongly associated with the degree of cerebral metabolite disturbance observed via MRS. Reduced cortical and hippocampal volumes were most strongly associated with decreased NAA, though reduced Glx also tended to be associated with reduced cortical and subcortical volumes (caudate and thalamus) as well, suggesting both neuronal and glial disturbances. Interestingly, metabolite-volumetric relationships were not limited to the cortical region from which MRS was measured, possibly reflecting shared pathophysiological processes. The relationships between Cho and volumetric measures suggest a complicated relationship possibly related to the effects of inflammatory processes on brain volume. The findings demonstrate the relationship between MRI-derived measures of cerebral metabolite disturbances and structural brain integrity, which has implication in understanding HIV-associated neuropathological mechanisms.

The basal ganglia communicate with the cerebellum.

The basal ganglia and cerebellum are major subcortical structures that influence not only movement, but putatively also cognition and affect. Both structures receive input from and send output to the cerebral cortex. Thus, the basal ganglia and cerebellum form multisynaptic loops with the cerebral cortex. Basal ganglia and cerebellar loops have been assumed to be anatomically separate and to perform distinct functional operations. We investigated whether there is any direct route for basal ganglia output to influence cerebellar function that is independent of the cerebral cortex. We injected rabies virus (RV) into selected regions of the cerebellar cortex in cebus monkeys and used retrograde transneuronal transport of the virus to determine the origin of multisynaptic inputs to the injection sites. We found that the subthalamic nucleus of the basal ganglia has a substantial disynaptic projection to the cerebellar cortex. This pathway provides a means for both normal and abnormal signals from the basal ganglia to influence cerebellar function. We previously showed that the dentate nucleus of the cerebellum has a disynaptic projection to an input stage of basal ganglia processing, the striatum. Taken together these results provide the anatomical substrate for substantial two-way communication between the basal ganglia and cerebellum. Thus, the two subcortical structures may be linked together to form an integrated functional network.

Neuroprotective activities of CEP-1347 in models of neuroAIDS.

When the nervous system is infected with HIV-1, it commonly results in neuroinflammation leading to overt neuronal dysfunction and subsequent cognitive and behavioral impairments. The multifaceted disease process, now referred to as HIV-1-associated neurocognitive disorders (HAND), provides a range of molecular targets for adjunctive therapies. One is CEP-1347, an inhibitor of mixed lineage kinases that elicits neuroprotective and anti-inflammatory responses in models of neurodegenerative diseases. Since HAND is associated with inflammatory encephalopathy induced by virus infection and mononuclear phagocytes (perivascular macrophages and microglia) immune activation, we investigated whether CEP-1347 could ameliorate disease in laboratory models of HAND. We now demonstrate that CEP-1347 reduces the levels of secreted proinflammatory cytokines and chemokines in HIV-1-infected human macrophages and attenuates dose-dependent neurotoxicity in rodent cortical neurons. CEP-1347-treated mice readily achieve therapeutic drug levels in peripheral blood. HIV-1 encephalitis (HIVE) mice, where human virus-infected monocyte-derived macrophages are stereotactically injected into the basal ganglia of CB17 severe combined immunodeficient mice, received daily intraperitoneal injections of CEP-1347. Here, CEP-1347 treatment of HIVE mice showed a dose-dependent reduction in microgliosis. Dendritic integrity and neuronal loss were sustained and prevented, respectively. These results demonstrate that CEP-1347 elicits anti-inflammatory and neuroprotective responses in an HIVE model of human disease and as such warrants further study as an adjunctive therapy for human disease.

Entacapone, a catechol-O-methyltransferase inhibitor, improves the motor activity and dopamine content of basal ganglia in a rat model of Parkinson's disease induced by Japanese encephalitis virus.

Levodopa is the main medication used for the treatment of Parkinson's disease. However, dyskinesia and wearing-off appear after the administration of high-dose levodopa for a long period. To combat the dyskinesia and wearing-off, levodopa is used together with a dopamine (DA) receptor agonist, and the amount of levodopa is decreased. We have reported the monoamine oxidase (MAO)-B inhibitor selegiline to be effective for treating motor dysfunction in Parkinson's disease model rats. We analyzed the improvement in motor functions and catecholamine contents in various brain regions induced by a combination of the catechol-O-methyltransferase (COMT) inhibitor entacapone and a levodopa/dopadecarboxylase inhibitor (DDCI) in Japanese encephalitis virus (JEV) induced Parkinson's disease model rats. Entacapone (10 mg/kg) was administered via a single oral administration with levodopa/DDCI (10 mg/kg). The motor functions of the JEV model rats were significantly worsened, compared with those of the healthy control rats. The motor functions in the Parkinson's disease model rats were significantly recovered to the same levels as the healthy control rats by the combined administration of entacapone and levodopa/DDCI. A significant improvement in motor function was not demonstrated in the case of the administration of levodopa/DDCI alone. The striatal DA concentrations in the model rat brains were significantly increased by using levodopa/DDCI together with entacapone. Motor function was recovered by raising the striatum DA density in the model rats. Thus, COMT inhibitors are useful for decreasing the amount of levodopa administered to Parkinson's disease patients.