Optimization of the fluid-attenuated inversion recovery (FLAIR) imaging for use in autopsy imaging of the brain region using synthetic MRI.

BACKGROUND: The failure of cerebrospinal fluid (CSF) signal suppression in postmortem fluid-attenuated inversion recovery (FLAIR) of the brain is a problem. OBJECTIVE: The present study was to clarify the relationship between the temperature of deceased persons and CSF T1, and to optimize the postmortem brain FLAIR imaging method using synthetic MRI. METHODS: Forehead temperature was measured in 15 deceased persons. Next, synthetic MRI of the brain was performed, the CSF T1 was measured, and the optimal TI was calculated. Two types of FLAIR images were obtained with the clinical and optimal TI. The relationship between forehead temperature and the CSF T1 and optimal TI was evaluated. The optimized FLAIR images were physically and visually evaluated. RESULTS: The CSF T1 and optimal TI were strongly correlated with forehead temperature. Comparing the average SNR and CNR ratios and visual evaluation scores of the two FLAIR images, those captured with the optimal TI showed statistically lower SNR, higher CNR, and higher visual evaluation scores (p< 0.01). CONCLUSIONS: Synthetic MRI enables the quantification of the CSF T1 resulting from postmortem temperature decreases and calculation of the optimal TI, which could aid in improving the failure of CSF signal suppression and in optimizing postmortem brain FLAIR imaging.

Elevation of inositol pyrophosphate IP7 in the mammalian spinal cord of amyotrophic lateral sclerosis.

Background: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder associated with progressive impairment of spinal motor neurons. Continuous research endeavor is underway to fully understand the molecular mechanisms associating with this disorder. Although several studies have implied the involvement of inositol pyrophosphate IP7 in ALS, there is no direct experimental evidence proving this notion. In this study, we analyzed inositol pyrophosphate IP7 and its precursor IP6 in the mouse and human ALS biological samples to directly assess whether IP7 level and/or its metabolism are altered in ALS disease state. Methods: We used a liquid chromatography-mass spectrometry (LC-MS) protocol originally-designed for mammalian IP6 and IP7 analysis. We measured the abundance of these molecules in the central nervous system (CNS) of ALS mouse model SOD1(G93A) transgenic (TG) mice as well as postmortem spinal cord of ALS patients. Cerebrospinal fluid (CSF) and peripheral blood mononuclear cells (PBMCs) from ALS patients were also analyzed to assess if IP7 status in these biofluids is associated with ALS disease state. Results: SOD1(G93A) TG mice showed significant increase of IP7 level in the spinal cord compared with control mice at the late stage of disease progression, while its level in cerebrum and cerebellum remains constant. We also observed significantly elevated IP7 level and its product-to-precursor ratio (IP7/IP6) in the postmortem spinal cord of ALS patients, suggesting enhanced enzymatic activity of IP7-synthesizing kinases in the human ALS spinal cord. In contrast, human CSF did not contain detectable level of IP6 and IP7, and neither the IP7 level nor the IP7/IP6 ratio in human PBMCs differentiated ALS patients from age-matched healthy individuals. Conclusion: By directly analyzing IP7 in the CNS of ALS mice and humans, the findings of this study provide direct evidence that IP7 level and/or the enzymatic activity of IP7-generating kinases IP6Ks are elevated in ALS spinal cord. On the other hand, this study also showed that IP7 is not suitable for biofluid-based ALS diagnosis. Further investigation is required to elucidate a role of IP7 in ALS pathology and utilize IP7 metabolism on the diagnostic application of ALS.

Formalin RT-QuIC assay detects prion-seeding activity in formalin-fixed brain samples from sporadic Creutzfeldt-Jakob disease patients.

BACKGROUND: The neuropathology of sporadic Creutzfeldt-Jakob disease (sCJD) is usually investigated using formalin-fixed and formic acid-treated brain tissue. However, formalin and formic acid treatment can interfere with immunostaining of abnormal prion protein. Therefore, there is a need for biochemical methods other than immunostaining to investigate abnormal prion protein in postmortem tissue. We developed RT-QuIC to quantitate the seeding activity (SD50) of sCJD brain tissue treated with formalin and formic acid. METHODS: We used endpoint RT-QuIC assays to analyze SD50 in formalin-fixed brain tissue from 19 sCJD patients (14 MM1 cases, 3 MM2-thalamic form [MM2T] cases and 2 MM2-cortical form [MM2C] cases) diagnosed according to Parchi's classification. We assessed SD50 in brains after incubation in formalin solution for over 1 month, and after treating formalin-fixed brain tissue with formic acid. We also examined how the SD50 values from formalin-fixed brain samples compared with neuropathological and immunohistochemical findings. RESULTS: The SD50 values of formalin-fixed brain samples from 14 MM1 cases, 2 MM2C cases, and 2 MM2T cases were 107.77±0.57/g tissue, 107.44±0.24/g tissue and 106.00±0.77/g tissue, respectively. The average SD50 value in MM1 unfixed brains decreased by 102.04 after formalin fixation for 1 month. In MM1 cases, after combined formalin and formic acid treatment, the SD50 value was reduced by approximately 105.16 compared with that of unfixed tissue. The SD50 values of formalin-fixed tissue showed a consistent pattern with the neuropathological findings in most brain regions examined. CONCLUSION: RT-QuIC enables the study of formalin-fixed brain tissue from sCJD patients that has not previously been amenable to analysis.

Inositol hexakisphosphate kinase 2 promotes cell death of anterior horn cells in the spinal cord of patients with amyotrophic lateral sclerosis.

We have previously reported that inositol hexakisphosphate kinase (InsP6K)2 mediates cell death. InsP6K2 is abundantly expressed in anterior horn cells of the mammalian spinal cord. We investigated the role of InsP6K2 in spinal cords of patients with amyotrophic lateral sclerosis (ALS). Autopsy specimens of lumbar spinal cords from ten patients with sporadic ALS and five non-neurological disease patients (NNDPs) were obtained. We performed quantitative real-time PCR, immunostaining, and western blotting for InsP6K1, InsP6K2, InsP6K3, protein kinase B (Akt), casein kinase 2 (CK2), and 90-kDa heat-shock protein (HSP90). In contrast to InsP6K1 and InsP6K3 mRNA expression, InsP6K2 levels in anterior horn cells of the spinal cord were significantly increased in ALS patients compared to NNDPs. In ALS patients, InsP6K2 translocated from the nucleus to the cytoplasm. However, we observed a decrease in HSP90, CK2, and Akt activity in ALS patients compared to NNDPs. A previous study reported that InsP6K2 activity is suppressed after binding to HSP90 and subsequent phosphorylation and degradation by CK2, thus decreasing InsP6K2 activity. However, InsP7, which is generated by InsP6K2, can compete with Akt for PH domain binding. Consequently, InsP7 can inhibit Akt phosphorylation. Our results suggest that InsP6K2 is activated in the spinal cord of patients with ALS and may play an important role in ALS by inducing cell death mechanisms via Akt, CK2, and HSP90 pathways.

Importance of Neuropathological Diagnosis of Dementia Patients in Family Practice.

INTRODUCTION: Creutzfeldt-Jakob disease (CJD) is an important dementia disorder. However, clinical diagnosis can be difficult and delayed for many primary physicians caring for dementia patients. The aim of the present study was to describe clinical and neuropathological results of an individual with CJD who was seen by a community hospital. Our report may inform many primary physicians on understanding the significance of CJD. METHODS: Clinical information was obtained from medical records. Neuropathological and biochemical analyses were performed using autopsied brain. RESULTS: A 58-year-old Japanese man who had worked as a carpenter developed memory and executive function impairments. He was initially diagnosed as having Alzheimer's disease based on clinical and neuroradiological analyses. Myoclonus was observed in the later stage of clinical course. Hyperintense lesions on diffusion-weighted images were observed in the cerebral cortex in later stage. Analysis of cerebrospinal fluid showed increased levels of total tau and phospho-tau protein. However, 14-3-3 protein and amyloid ß (1-42) were normal. Genetic analysis of the PRNP gene showed methionine homozygosity at codon 129 and glutamate homozygosity at codon 219. The results of neuropathological analysis were consistent with sporadic CJD (MM2 cortical type with some type 1 pattern of 3F4 immunoreactivity). Western blot analysis of the frontal and cerebellar cortex revealed a type 2 and type 1 pattern of proteinase K (PK)-resistant prion protein, respectively. No Alzheimer's pathology was present. CONCLUSIONS: Our experience may help primary physicians to assess dementia patients. Since atypical forms of prion disease are now well-established, we need to consider prion disease in dementia patients. Clinical examination alone is not enough for dementia workup; thus, we must understand the importance of neuropathological study and encourage autopsy to reach a definite diagnosis of dementia.

Cerebral Microbleeds Detected Using 3.0T Magnetic Resonance Imaging in 2,003 Patients with Ischemic or Hemorrhagic Stroke.

INTRODUCTION: Compared with 1.5T magnetic resonance imaging (MRI), using 3.0T MRI makes it easier to detect cerebral microbleeds (CMBs). We used 3.0T MRI to investigate the backgrounds, risk factors, and number and location of CMBs in patients with ischemic or hemorrhagic stroke. METHODS: We extracted data on clinical characteristics, risk factors, and number and location of CMBs in 2,003 patients treated between January 2010 and December 2014 within one week of stroke occurrence. We then carried out multivariate analysis of the data. RESULTS: CMBs were present in 1,025 patients. The numbers of CMBs in ischemic stroke and hemorrhagic stroke patients were 9,410 and 6,419, respectively. Patients with CMBs showed significantly higher rates of cognitive impairment (p < 0.001, odds ratio [OR] = 1.514), hypertension (p < 0.001, OR = 3.145), previous history of stroke (p < 0.001, OR = 1.782), and presence of hemorrhagic stroke (p < 0.001, OR = 2.066). The use of antithrombotic medication before the stroke did not affect the incidence of CMBs. In ischemic stroke patients, patients with small vessel occlusion had a significantly greater rate of previous history of hemorrhagic stroke (p = 0.046) and number of patients with CMBs (p < 0.001) than those with cardioembolism. CONCLUSIONS: CMBs were well observed in patients with small vessel disease, and hypertension was an important factor in ischemic and hemorrhagic stroke. Antithrombotic medication is not associated with the development of CMBs if adequate antihypertensive therapy is provided.

Postmortem Quantitative Analysis of Prion Seeding Activity in the Digestive System.

Human prion diseases are neurodegenerative disorders caused by prion protein. Although infectivity was historically detected only in the central nervous system and lymphoreticular tissues of patients with sporadic Creutzfeldt-Jakob disease, recent reports suggest that the seeding activity of Creutzfeldt-Jakob disease prions accumulates in various non-neuronal organs including the liver, kidney, and skin. Therefore, we reanalyzed autopsy samples collected from patients with sporadic and genetic human prion diseases and found that seeding activity exists in almost all digestive organs. Unexpectedly, activity in the esophagus reached a level of prion seeding activity close to that in the central nervous system in some CJD patients, indicating that the safety of endoscopic examinations should be reconsidered.

De Novo Arteriovenous Malformation after Aneurysm Clipping.

We report a 73-year-old woman with de novo arteriovenous malformations (AVMs) that developed in the ipsilateral parietal lobe after craniotomy and aneurysm clipping. While intracerebral AVMs are considered to be congenital lesions, there have been several reported cases of acquired AVM arising after ischemic or traumatic episodes. We summarize previously reported cases of such acquired 'de novo' AVMs with a discussion of some pathophysiological responses or factors suggested to promote their development.

Pathologic Study of Intracranial Large Artery Atherosclerosis in 7260 Autopsy Cases.

BACKGROUND: Atherosclerotic changes in the cerebral arteries may differ with era of birth. Herein, we analyzed the chronological changes of intracranial atherosclerosis in consecutive autopsy cases. METHODS: A total of 7260 autopsy cases from 1972 to 2014 were analyzed. Severity of atherosclerosis was classified using a semi-quantitative scale of pathologic observation of each artery after formalin fixation: 0 = no stenosis; .5 = fatty streaks but no stenosis; 1 = <50% stenosis; 2 = 50%-90% stenosis; 3 = ≥90% stenosis. The bilateral vertebral, anterior, middle, and posterior cerebral arteries and the basilar artery were scored. The sum of each individual was defined and compared by age at death, sex, and era of birth. RESULTS: The atherosclerosis score increased with age at death, as follows: age in the 50s, 0 [0-2]; 60s, 3 [.5-7]; 70s, 5 [2-9.5]; 80s, 6.5 [3.5-11.5]; 90s, 7.75 [4-12]; and 100s, 8 [5.5-13.5] (median value [interquartile range], P< .0001). The percentage of cases with a score of 2 or 3 in each artery also increased with age (P< .0001). Atherosclerosis score was higher in men than women in their 60s at death, and was higher in women than men in their 80s and 90s at death. In each age at death group (from 60s to 100s), the score declined with later year of birth (P < .05). CONCLUSIONS: Intracranial atherosclerosis advances with age and is more severe in subjects born earlier.

Identification of hemodynamically compromised regions by means of cerebral blood volume mapping utilizing computed tomography perfusion imaging.

The aim of the study was to evaluate the potential role of computed tomography perfusion (CTP) imaging in identifying hemodynamically compromised regions in patients with occlusive cerebrovascular disease. Twelve patients diagnosed with either occlusion or severe stenosis of the internal carotid artery or the M1 portion of the middle cerebral artery underwent CTP imaging. The data was analyzed by an automated ROI-determining software. Patients were classified into two subgroups: an asymptomatic group consisting of three patients in whom perfusion pressure distal to the site of occlusion/stenosis (PPdis) could be maintained in spite of the arterial occlusion/stenosis, and a symptomatic group consisting of nine patients in whom PPdis could not be maintained enough to avoid watershed infarction. Four CTP-related parameters were independently compared between the two groups. Significant differences were determined using a two-sample t-test. When statistically significant differences were identified, cut-off points were calculated using ROC curves. Analysis revealed statistically significant differences between the asymptomatic and symptomatic subgroups only in the measure of relCBV (p=0.028). Higher relCBV values were observed in the symptomatic subgroup. ROC curve analysis revealed 1.059 to be the optimal relCBV cut-off value for distinguishing between the asymptomatic and symptomatic subgroups. The data revealed that, in patients whose PPdis is maintained, relCBV remains around 1.00. Conversely, in patients whose PPdis decreased, relCBV increased. From these findings, we conclude that elevation of relCBV as observed using CTP imaging accurately reflects the extent of compensatory vasodilatation involvement and can identify hemodynamically compromised regions.

Neuropathology of supercentenarians - four autopsy case studies.

Supercentenarians (aged 110 years old or more) are extremely rare in the world population (the number of living supercentenarians is estimated as 47 in the world), and details about their neuropathological information are limited. Based on previous studies, centenarians (aged 100-109 years old) exhibit several types of neuropathological changes, such as Alzheimer's disease and Lewy body disease pathology, primary age-related tauopathy, TDP-43 pathology, and hippocampal sclerosis. In the present study, we provide results from neuropathological analyses of four supercentenarian autopsy cases using conventional and immunohistochemical analysis for neurodegenerative disorders. In particular, we focused on the pathology of Alzheimer's disease and Lewy body disease, as well as the status of hippocampal sclerosis, TDP-43 pathology, aging-related tau astrogliopathy, and cerebrovascular diseases. Three cases were characterized as an "intermediate" level of Alzheimer's disease changes (NIA-AA guideline) and one was characterized as primary age-related tauopathy. TDP-43 deposits were present in the hippocampus in two cases. Neither Lewy body pathology nor hippocampal sclerosis was observed. Aging-related tau astrogliopathy was consistently observed, particularly in the basal forebrain. Small vessel diseases were also present, but they were relatively mild for cerebral amyloid-beta angiopathy and arteriolosclerosis. Although our study involved a small number of cases, the results provide a better understanding about human longevity. Neuropathological alterations associated with aging were mild to moderate in the supercentenarian brain, suggesting that these individuals might have some neuroprotective factors against aging. Future prospective studies and extensive molecular analyses are needed to determine the mechanisms of human longevity.

Prion-Seeding Activity Is widely Distributed in Tissues of Sporadic Creutzfeldt-Jakob Disease Patients.

Human prion diseases are neurodegenerative disorders caused by abnormally folded prion proteins in the central nervous system. These proteins can be detected using the quaking-induced conversion assay. Compared with other bioassays, this assay is extremely sensitive and was used in the present study to determine prion distribution in sporadic Creutzfeldt-Jakob disease patients at autopsy. Although infectivity of the sporadic form is thought to be restricted within the central nervous system, results showed that prion-seeding activities reach 106/g from a 50% seeding dose in non-neuronal tissues, suggesting that prion-seeding activity exists in non-neural organs, and we suggested that non-neural tissues of 106/g SD50 did not exist the infectivity.

Characteristics of subcortical infarction due to distal MCA penetrating artery occlusion.

OBJECTIVE: Isolated deep subcortical infarcts develop as a result of occlusion of the penetrating arteries from the internal carotid artery (ICA) and the proximal (M1) and distal middle cerebral artery (MCA). However, the clinical and neuroimaging characteristics of infarcts due to the occlusion of the distal MCA penetrating artery are unclear. METHODS: Consecutive patients with ischemic stroke or transient ischemic attack with magnetic resonance imaging (MRI) performed within 2days of onset were studied retrospectively. Using coronal MRI data, isolated deep subcortical infarcts were classified into two groups: 1) proximal group, described as being longer than they are wide, which were expected to be related to the occlusion of the ICA or M1 penetrating artery; and 2) distal group, described as oblong, which were expected to be associated with the occlusion of penetrating arteries from the distal MCA (M2/M3/M4). RESULTS: A total of 653 consecutive acute ischemic stroke patients (proximal group, 50 [7.7%]; distal group, 14 [2.1%]) were enrolled. Baseline clinical characteristics were not different between the 2 groups. Modified Rankin Scale scores were lower in the distal group than in the proximal group 3months after stroke onset (1.43±0.36 vs. 2.26±1.35, p=0.023). We measured the lengths of the infarcts in the X and Y directions using axial MRI. The X/Y ratio was larger in the distal group than in the proximal group (1.3±0.6 vs. 0.7±0.2, p<0.01), which indicated that distal MCA penetrating artery infarcts appear more oblong on axial MRI. CONCLUSIONS: One cause for deep subcortical infarction is the occlusion of the distal MCA penetrating arteries, which occurs in 22% of patients with deep subcortical infarctions. These patients had better clinical outcomes than those with ICA and M1 penetrating artery infarctions. Distal MCA penetrating artery infarctions appear oblong on axial MRI.

Changes in intracortical inhibition and clinical symptoms after STN-DBS in Parkinson's disease.

OBJECTIVES: To examine effects of subthalamic nucleus deep brain stimulation (STN-DBS) on intracortical inhibition in Parkinson's disease (PD) and the correlation between intracortical inhibition and clinical symptoms after alteration of STN-DBS status. METHODS: Nine PD patients treated by STN-DBS were compared with eight age-matched controls. Antiparkinsonian medication was withdrawn 12h before the study. Short-interval intracortical inhibition (SICI) with a 3-ms interval and silent period (SP) were examined using transcranial magnetic stimulation. SP duration, SICI and motor symptoms (rigidity and tremor) were evaluated with STN-DBS ON, and over 120 min during STN-DBS OFF. RESULTS: Even during STN-DBS, PD patients showed a shortened SP and reduced SICI relative to normal controls. SICI decreased further throughout STN-DBS OFF, resulting in facilitation rather than inhibition; SP shortened only after 120 min STN-DBS OFF. Both rigidity and tremor worsened throughout STN-DBS OFF, with a time course similar to SICI. CONCLUSION: Even during STN-DBS, both SICI and SP in PD patients remain impaired without medication. Changes in SICI, but not SP, show a time course similar to those of motor symptoms. SIGNIFICANCE: The dissimilarity of SICI and SP changes suggests differences in mediation of inhibitory mechanisms and/or superimposition of exaggerated intracortical facilitation on SICI.

Correlation between the Occlusion Site and Clinical Outcome after Acute Ischemic Stroke.

OBJECTIVE: The significance of early mild clinical improvement after intravenous tissue plasminogen activator (IVtPA) treatment is unclear. Therefore, we examined whether the timing of clinical improvement after IVtPA predicted the clinical outcome at 3 months. METHODS: Consecutive patients with acute cerebral infarction in the anterior circulation who received IVtPA treatment within 3 hours of the onset of symptoms were enrolled in the study. Patients were classified according to the timing of clinical improvement [early responder (ER), National Institutes of Health Stroke Scale (NIHSS) score improved ≥4 points or who had a score of 0 within 2 hours after IVtPA; late responder (LR), a similar improvement between 2 and 24 hours; and non-responder (NR)] and according to the arterial occlusion site (P group, internal carotid artery and proximal middle cerebral artery M1 region; and D group, distal M1 and M2). RESULTS: Ninety-three patients [median age, 74 (67-79) years; 54 men (58%); median NIHSS score, 11 (7-16)] were enrolled in the study. The P group consisted of 48 (52%) patients and the D group consisted of 45 (48%) patients. Thirty-eight patients (41%) were classified as ERs, 20 (22%) as LRs, and 35 (38%) as NRs. On a multivariate regression analysis, the P group [odds ratio (OR), 3.24; 95% confidence interval (CI), 1.08-10.45; p=0.036] and NR (OR, 4.04; 95% CI, 1.29-14.27; p=0.016) were independent predictors of a poor outcome. ER (47%, p=0.01) and LR (45%, p=0.01) patients showed fewer poor outcomes than NR (77%) patients, but the rate did not differ significantly between the ER and LR patients. CONCLUSION: Early mild clinical recovery did not predict a good outcome. The occlusion site was a stronger predictor of clinical improvement after IVtPA administration.

Hemodynamic stress distribution reflects ischemic clinical symptoms of patients with moyamoya disease.

OBJECTIVE: Currently, the probability of diagnosing asymptomatic moyamoya disease is increasing. In this study, we consider a less invasive method for predicting future ischemic symptoms in patients with moyamoya disease. METHODS: We reviewed cerebral blood flow (CBF)-related data obtained by xenon CT imaging (XeCT) in six patients with ischemic-type or asymptomatic moyamoya disease. The data were obtained as volume data using a 320-row CT, and applied to the automated region-of-interest-determining software (3DSRT) and converted to standardized images. Eight CBF-related parameters, including CBF value, cerebrovascular reserve capacity (CVRC), and hemodynamic distribution (hdSD), were compared between asymptomatic hemispheres and ischemic symptomatic hemispheres. A significant difference was determined by a two-sample t test. A difference with p<0.05 was considered significant. When statistically significant differences between parameters of asymptomatic hemispheres and ischemic symptomatic hemispheres were identified, cut-off points were calculated with receiver operating characteristic (ROC) curves. Change in the parameters before and after bypass surgery was also assessed. RESULTS: Of the eight CBF-related parameters evaluated, statistically significant differences between the asymptomatic hemispheres and ischemic hemispheres were observed in the CBF value of the MCA region (CBF-MCA), both at rest and after acetazolamide loading, and in the hdSD, also both at rest and after acetazolamide loading. Of the four statistically significant parameters, ROC analysis revealed that the hdSD at rest and CBF-MCA after acetazolamide loading were the most sensitive and specific parameters (threshold 1.2, sensitivity 1, specificity 1 for hdSD at rest, and threshold 26.44mL/100g/min, sensitivity 1, specificity 1 for CBF-MCA after acetazolamide loading). From the CBF data obtained both before and after surgery from the three patients who had undergone direct bypass surgeries, the hdSD was higher than the threshold of 1.2 before surgery but decreased to lower than the threshold of 1.2 after surgery. Ischemic symptoms also resolved after surgery. CONCLUSIONS: The data showed that hdSD at rest and CBF-MCA after acetazolamide loading reflects ischemic symptoms of patients with moyamoya disease. Thus, these parameters could be used as ischemic symptom markers for following patients with moyamoya disease. hdSD at rest is important because it is less invasive and can be performed without acetazolamide loading.

Rapid and Quantitative Assay of Amyloid-Seeding Activity in Human Brains Affected with Prion Diseases.

The infectious agents of the transmissible spongiform encephalopathies are composed of amyloidogenic prion protein, PrPSc. Real-time quaking-induced conversion can amplify very small amounts of PrPSc seeds in tissues/body fluids of patients or animals. Using this in vitro PrP-amyloid amplification assay, we quantitated the seeding activity of affected human brains. End-point assay using serially diluted brain homogenates of sporadic Creutzfeldt-Jakob disease patients demonstrated that 50% seeding dose (SD50) is reached approximately 10(10)/g brain (values varies 10(8.79-10.63)/g). A genetic case (GSS-P102L) yielded a similar level of seeding activity in an autopsy brain sample. The range of PrPSc concentrations in the samples, determined by dot-blot assay, was 0.6-5.4 µg/g brain; therefore, we estimated that 1 SD50 unit was equivalent to 0.06-0.27 fg of PrPSc. The SD50 values of the affected brains dropped more than three orders of magnitude after autoclaving at 121°C. This new method for quantitation of human prion activity provides a new way to reduce the risk of iatrogenic prion transmission.