Involvement of neuronal nitric oxide synthase in cognitive impairment in isoflurane-treated rats

The underlying causes of post-operative cognitive dysfunction (POCD) in elderly patients remain to be elucidated. In order to explore possible contributory mechanisms, we tested the effects of isoflurane anesthesia on (i) expression of hippocampal neuronal nitric oxide synthase (nNOS) and (ii) the relationship of changes in nNOS expression to cognitive dysfunction in isoflurane-treated aged rats. Our results indicate that isoflurane treatment leads to significant changes in correct reactions (F = 28.35, p < 0.001), initiative avoidances (F = 29.33, p < 0.001), and total reaction time (TRT) (F = 6.99, p < 0.05) of treated rats in the Y-maze test. Isoflurane-treated rats had fewer correct reactions and initiative avoidances in the Y-maze test 24 and 48 h after 2 h of isoflurane anesthesia compared with control group rats (p < 0.05). TRTs to complete 20 trials of the Y-maze test increased significantly 48 h after 2 h anesthesia. The number of nNOS-positive hippocampal neurons decreased 24 h after anesthesia, corresponding to an increased mean immunostaining grey-scale value. These data show that isoflurane causes a transient decrease in expression of hippocampal nNOS in aged rats during early post-anesthesia stages, and that the transient decrease of nNOS is closely correlated with cognitive impairment in isoflurane-treated aged rats.     

Identification of Focal Epileptogenic Networks in Generalized Epilepsy Using Brain Functional Connectivity Analysis of Bilateral Intracranial EEG Signals

Simultaneous bilateral onset and bi-synchrony epileptiform discharges in electroencephalogram (EEG) remain hallmarks for generalized seizures. However, the possibility of an epileptogenic focus triggering rapidly generalized epileptiform discharges has been documented in several studies. Previously, a new multi-stage surgical procedure using bilateral intracranial EEG (iEEG) prior to and post complete corpus callosotomy (CC) was developed to uncover seizure focus in non-lateralizing focal epilepsy. Five patients with drug-resistant generalized epilepsy who underwent this procedure were included in the study. Their bilateral iEEG findings prior to complete CC showed generalized epileptiform discharges with no clear lateralization. Nonetheless, the bilateral ictal iEEG findings post complete CC indicated lateralized or localized seizure onset. This study hypothesized that brain functional connectivity analysis, applied to the pre CC bilateral iEEG recordings, could help identify focal epileptogenic networks in generalized epilepsy. The results indicated that despite diffuse epileptiform discharges, focal features can still be observed in apparent generalized seizures through brain connectivity analysis. The seizure onset localization/lateralization from connectivity analysis demonstrated a good agreement with the bilateral iEEG findings post complete CC and final surgical outcomes. Our study supports the role of focal epileptic networks in generalized seizures.     

Histological evaluation of patients with gastritis at high risk of developing gastric cancer using a conventional index

Although gastric cancer (GCa) is strongly associated with Helicobacter pylori infection, only some H. pylori-positive subjects develop gastric cancer. The aim of this study is to identify H. pylori-positive subjects at high risk of developing GCa by assessment of the histopathological findings in the non-cancer-containing mucosa of patients with and without GCa.The subjects were 35 patients with diffuse-type gastric cancer (D-GCa), 55 with intestinal-type gastric cancer (I-GCa), and 99 H. pylori-positive controls without GCa. Two specimens were taken from the greater curvature of the antrum and the middle body. Histopathological gradings were evaluated using the updated Sydney System, and the risk of GCa was evaluated using a modified Meining's gastric cancer risk index (GCRI). Among the H. pylori-positive controls, corpus gastritis was seen in 98.0% (97/99) and corpus atrophic gastritis in 78.8% (78/99). The mean GCRI for the D-GCa (5.514 ± 2.03) and I-GCa (6.836 ± 2.08) groups was significantly greater than that for the H. pylori-positive controls (4.071 ± 2.07; p = 0.0005, p < 0.0001). In addition, the mean GCRI for the I-GCa group was significantly greater than that for the D-GCa group (p < 0.005). The GCRI-positive rate was significantly higher in subjects with GCa than in H. pylori-positive controls (D-GCa: p < 0.005, I-GCa: p < 0.0001).Many H. pylori-positive Japanese still carry a high risk for gastric cancer. However, H. pylori-positive patients at high risk of developing GCa (not only intestinal-type but also diffuse-type) may be detected using a simple GCRI.     

B7-H1 and B7-DC receptors of oral squamous carcinoma cells are upregulated by Porphyromonas gingivalis

The up-regulation of the B7-H1 receptors in host cells might influence the chronicity of inflammatory disorders that frequently precede the development of human cancers. B7-H1 expression has been detected in the majority of human cancers, leading to anergy and apoptosis of activated T cells, and enabling tumor cells to overcome host response. Porphyromonas gingivalis (P. gingivalis), a putative periodontal pathogen, is an etiologic agent of periodontitis and expresses a variety of virulence factors. In this study, the expression of B7-H1 and B7-DC receptors on squamous cell carcinoma cells SCC-25 and BHY and primary human gingival keratinocytes (PHGK) was analyzed after infection with two virulent P. gingivalis strains in vitro. After 48 h, the cells were stained with antibodies for human B7-H1 and B7-DC and further analyzed by flow cytometry. RNA was extracted and gene expression of B7-H1 or B7-DC was quantified by real time PCR. After infection with P. gingivalis, both B7-H1 and B7-DC receptors were up-regulated.The mean fluorescence intensity (MFI) increased from 4.5 to 9.9 (B7-H1) and from 6.9 to 15.0 (B7-DC) (p < 0.05, respectively) in SCC-25 cells. PHGK showed an increase from 4.8 to 12.4 (B7-H1) and from 5.5 to 15.6 (B7-DC) (p < 0.05, respectively). Streptococcus salivarius K12, a commensal bacterium, caused no up-regulation. After 24 h, the expression of B7H1 and B7-DC mRNA in infected cells, normalized to GAPDH and in relation to non-infected cells, was 6.4 fold (B7-H1) and 8.6 fold (B7-DC) higher. In PHGK B7-H1/DC mRNA expression increased 8.2 fold (B7-H1) and 5.9 fold (B7DC) (p < 0.05) respectively. The results of the study demonstrate that in contrast to S. salivarius K12 virulent P. gingivalis strains are able to induce the expression of the B7-H1 and B7-DC receptors in squamous carcinoma cells and human gingival keratinocytes, which might facilitate immune evasion by oral cancers.     

Circulating IL-35 in pancreatic ductal adenocarcinoma patients

IL-35 is a novel inhibitory cytokine that is mainly produced by regulatory T-cells (Tregs) and is required for Treg-mediated immunosuppression. However, the plasma levels of IL-35 in patients with pancreatic ductal adenocarcinoma (PDAC) have never been investigated. In this study, we found that plasma IL-35 levels more significantly increased in PDAC patients than in normal controls (134.53 ± 92.45 pg/mL vs. 14.26 ± 6.56 pg/mL). IL-35 mRNA levels were positively correlated with plasma IL-35 levels (EBI3, R = 0.925, p < 0.01; p35, R = 0.916, p < 0.01). Furthermore, IL-35 expression levels were associated with lymph node metastasis (p = 0.001) and late tumor stage (p = 0.002). For the resected patients, high IL-35 expression levels were associated with large tumor size (p < 0.01), higher TNM classification T staging (p < 0.05), and late tumor stage (p < 0.05). In conclusion, circulating IL-35 in PDAC patients significantly increased, suggesting that regulating the expression of IL-35 may provide a new possible target for the treatment of PDAC patients, especially for the resectable ones.     

Circulating levels of GDNF in bipolar disorder

Neurotrophic factors regulate the survival and growth of neurons, and influence synaptic efficiency and plasticity. Several studies suggest the existence of a relationship between changes in neurotrophic levels and bipolar disorder (BD). The glial cell-line derived neurotrophic factor (GDNF) influences monoaminergic neurons and glial cells, but its role in BD patients is controversial. In order to elucidate it we evaluated plasma levels of GDNF in a sample of 70 BD patients (35 in mania and 35 in euthymia) and compared with 50 healthy controls matched for age, gender and educational levels. GDNF plasma levels were measured by enzyme-linked immunosorbent assay (ELISA). Patients were assessed by a Mini-International Neuropsychiatric Interview (MINI-plus), Young Mania and Hamilton Depression Rating Scales. Plasma GDNF levels were significantly increased in BD patients in euthymia compared with BD patients in mania and healthy controls (p < 0.05). GDNF plasma levels were correlated with age (ρ = 0.30, p < 0.05) and negatively correlated with manic symptoms in BD patients (ρ = −0.54, p < 0.05). Our results provide evidence that peripheral levels of GDNF are related with different mood states in BD, reinforcing the involvement of neurotrophic factors in its physiopathology.     

Differential response of AMPA and NMDA glutamate receptors of Purkinje cells to aging of the chicken cerebellum

Aging can lead to cognitive, affective, learning, memory and motor deficits. Since the cerebellum and glutamatergic neurotransmission are involved in several of those functions, the present work aimed at studying the expression of AMPA and NMDA glutamate receptor subunits in the chick cerebellum during aging. Young (30 days old) and aged (ca. 4 years old) chickens (Gallus gallus) were used in order to evaluate the expression of GluR1, GluR2/3 and NR1 subunits. The cerebella of young and aged chickens were subjected to immunohistochemical and immunoblotting techniques. Numbers of GluR1, GluR2/3 and NR1-positive cells and optical density of the immunoblotting data were analyzed and submitted to statistical analysis using ANOVA and the Bonferroni post hoc test. Mean density of Purkinje cells stained for Giemsa, GluR1, GluR2/3 and NR1 in the cerebellum all showed a statistically significant decrease in aged animals when compared to the young animals (Giemsa, P < 0.01; GluRs and NR1, P < 0.03). However, the ratio of GluR1 and GluR2/3-positive Purkinje cells in relation the total number of Purkinje cells found in each time point decreased with aging (ca. 10%), whereas the ratio of NR1-positive cells increased (ca. 9%). The immunoblotting data showed a significant decrease of GluR1 (ca. 66%) and GluR2/3 (ca. 55%) protein expression with aging, but did not reveal changes for NR1. Our data suggest that aging can lead to differential changes in the pattern of expression of glutamate receptor subunits, which can underlie at least part of the cognitive and motor disorders found in aged animals.     

Low-frequency stimulation of the hippocampal CA3 subfield is anti-epileptogenic and anti-ictogenic in rat amygdaloid kindling model of epilepsy

Neuromodulation with low-frequency stimulation (LFS), of brain structures other than epileptic foci, is effective in inhibiting seizures in animals and patients, whereas selection of targets for LFS requires further investigation. The hippocampal CA₃ subfield is a key site in the circuit of seizure generation and propagation. The present study aimed to illustrate the effects of LFS of the CA₃ region on seizure acquisition and generalization in the rat amygdaloid kindling model of epilepsy. We found that LFS (monophasic square-wave pulses, 1 Hz, 100 μA and 0.1 ms per pulse) of the CA₃ region significantly depressed the duration of epileptiform activity and seizure acquisition by retarding progression from focal to generalized seizures (GS). Moreover, GS duration was significantly shortened and its latency was significantly increased in the LFS group demonstrating an inhibition of the severity of GS and the spread of epileptiform activity. Furthermore, LFS prevented the decline of afterdischarge threshold (ADT) and elevated GS threshold indicating an inhibition of susceptibility to GS. These results suggest that LFS of the hippocampal CA₃ subfield is anti-epileptogenic and anti-ictogenic. Neuromodulation of CA₃ activity using LFS may be an alternative potential approach for temporal lobe epilepsy treatment.     

Diuretic-induced hyponatremia and osteoporotic fractures in patients admitted to the emergency department

Objective

Hyponatremia is a complication of diuretic treatment and has been recently identified as a novel factor associated with osteoporosis and fractures. The impact of diuretic-associated electrolyte disorders on osteoporotic fractures (OF) has rarely been studied systematically.

Design and setting

We conducted a study in patients presenting to the emergency department at the University Hospital Bern. In this retrospective case series analysis of prospectively gathered data, over a 2-year period we identified 10,823 adult (≥50 years) outpatients with a measured baseline serum sodium, at admission to the hospital. OF patients were compared to a control group without fractures using standard statistical methods.

Results

Four hundred and eighty (5%) patients had 547 OF. The OF group had a higher mean age (73 vs. 68 years, p < 0.0001), smaller proportion of men (37% vs. 58%, p < 0.0001), higher hospitalisation rate (83% vs. 62%, p < 0.0001) and longer hospital stay (8 vs. 6 days, p < 0.0001). Any diuretic agent (p < 0.0001), loop diurietics (p = 0.02), spironolactone (p = 0.02) and amiloride (p < 0.01) were used significantly more in OF patients, but not thiazides (p = 0.68). The prevalence of hyponatremia increased significantly (p < 0.0001) with the number of diuretics taken. Advanced age (odds ratio [OR] 1.04, p < 0.0001), hyponatremia (OR 1.46, p = 0.011) higher serum creatinine (OR 1.53, p = 0.0001), furosemide use alone (OR 1.40, p = 0.01) and co-treatment with amiloride (OR 2.22, p = 0.02) were associated with a higher risk for OF.

Conclusions

This study highlights the clinical association of hyponatremia during the use of certain diuretics (i.e. furosemide or in combination, i.e. amiloride) with an increased risk of osteoporosis associated fractures. Although evidence-based data is currently lacking a pragmatic approach concerning hyponatremia monitoring and correction appears reasonable in selected groups of patients.     

Down-regulation of aminolevulinate synthase,the rate-limiting enzyme for heme biosynthesis in Alzheimer's disease

Heme is an essential cell metabolite, intracellular regulatory molecule, and protein prosthetic group. Given the known alterations in heme metabolism and redox metal distribution and the up-regulation of heme oxygenase enzyme in Alzheimer's disease (AD), we hypothesized that heme dyshomeostasis plays a key role in the pathogenesis. To begin testing this hypothesis, we used qRT-PCR to quantify the expression of aminolevulinate synthase (ALAS1) and porphobilinogen deaminase (PBGD), rate-limiting enzymes in the heme biosynthesis pathway. The relative expression of ALAS1 mRNA, the first and rate-limiting enzyme for heme biosynthesis under normal physiological conditions, was significantly (p < 0.05) reduced by nearly 90% in AD compared to control. Coordinately, the relative expression of PBGD mRNA, which encodes porphobilinogen deaminase, the third enzyme in the heme synthesis pathway and a secondary rate-limiting enzyme in heme biosynthesis, was also significantly (p < 0.02) reduced by nearly 60% in AD brain compared to control and significantly related to apolipoprotein E genotype (p < 0.005). In contrast, the relative expression of ALAD mRNA, which encodes aminolevulinate dehydratase, the second and a non-rate-limiting enzyme for heme biosynthesis, was unchanged between the two groups. Taken together, our results suggest regulation of cerebral heme biosynthesis is profoundly altered in AD and may contribute toward disease pathogenesis by affecting cell metabolism as well as iron homeostasis.     

Anticholinesterase-induced epileptiform activity in immature rat piriform cortex slices, in vitro

Purpose: Acute in vitro brain slice models are commonly used to study epileptiform seizure generation and to test anti-epileptic drug action. Seizure-like activity can be readily induced by manipulating external ionic concentrations or by adding convulsant agents to the bathing medium. We previously showed that epileptiform bursting was induced in slices of immature (P14–28) rat piriform cortex (PC) by applying oxotremorine-M, a potent muscarinic receptor agonist. Here, we examined whether raising levels of endogenous acetylcholine (ACh) by exposure to anticholinesterases, could also induce epileptiform events in immature (P12–14) or early postnatal (P7–9) rat PC brain slices. Methods: The effects of anticholinesterases were investigated in rat PC neurons using both extracellular MEA (P7–9 slices) and intracellular (P12–14 slices) recording methods. Results: In P7–9 slices, eserine (20 μM) or neostigmine (20 μM) induced low amplitude, low frequency bursting activity in all three PC cell layers (I–III), particularly layer III, where neuronal muscarinic responsiveness is known to predominate. In P12–14 neurons, neostigmine produced a slow depolarization together with an increase in input resistance and evoked cell firing. Depolarizing postsynaptic potentials evoked by intrinsic fibre stimulation were selectively depressed although spontaneous bursting was not observed. Neostigmine effects were blocked by atropine (1 μM), confirming their muscarinic nature. We conclude that elevation of endogenous ACh by anticholinesterases can induce bursting in early postnatal PC brain slices, further highlighting the epileptogenic capacity of this brain region. However, this tendency declines with further development, possibly as local inhibitory circuit mechanisms become more dominant.     

64导长程视频脑电图对癫癎与发作性疾病的监测价值

目的：探讨64导长程视频脑电图（64-Channel Long-term video-EEG）对癫癎的诊断及鉴别诊断价值。方法：186例常规EEG无癎样放电患者分为临床癫癎组和可疑癫癎组进行长程VEEG监测并分析结果。结果：临床癫癎组癎样放电总发生率为71.4%,可疑癫癎组总发生率为32.2%。癎样放电最多出现于非快速眼动睡眠Ⅰ、Ⅱ期。各种发作类型癎样放电阳性率无统计学差异（P〉0.05）。结论：长程VEEG有利于提高癎样放电的检出率,对癫癎的诊断与分型具重要意义。     

257例癫癎与发作性疾病的24h动态脑电图监测

目的 :探讨动态脑电图 (AEEG ,即脑Holter)对癫的诊断及鉴别诊断价值。方法 :2 5 7例常规EEG无样放电患者分为临床癫组和可疑癫组进行 2 4hAEEG监测并分析结果。结果 :临床癫组样放电总发生率为 5 9.4 % ,可疑癫组总发生率为 19.9%。样放电最多出现于非快速眼动睡眠期。各种发作类型样放电阳性率差别无显著性。结论 :AEEG可提高样放电的检出率 ,对癫的诊断有重要意义     

动态脑电图对癫痫诊断和鉴别诊断的意义

对临床诊断癫痫的５３例和可疑癫痫的１１７例行２４ｈ动态脑电图（ＡＥＥＧ）监测。结果ＡＥＥＧ阳性率（３２．９％）明显高于常规ＥＥＧ（２０％），ＡＥＥＧ痫样放电率在癫痫组为５４．７％（２９／５３），可疑癫痫组为１５．４％（１８／１１７）。后者中有临床发作者６３例，检出痫样放电１０例，睡眠期出现痫样放电占８９．４％（４２／４７）。本文对痫样放电出现与临床发作间的关系以及癫痫诊断和鉴别诊断进行了讨论。     

Chronic treatment with ascorbic acid enhances cortical spreading depression in developing well-nourished and malnourished rats

Ascorbic acid (AA) is an antioxidant molecule that is highly concentrated in the brain and can exert both anticonvulsant and proconvulsant effects in distinct models of experimental seizures. Herein, we investigated whether chronic AA administration alters cortical excitability as indexed by the cortical spreading depression (CSD). Well-nourished (W) and malnourished (M) rats were treated, by gavage, with 60 mg/kg/day of l-AA from postnatal days 7–28, and CSD propagation was analyzed at 30–40 days. Compared to the W groups, M rats presented higher (p < 0.05) CSD amplitudes and velocities of propagation. In both nutritional conditions, AA-treatment significantly increased CSD amplitudes and propagation velocities (p < 0.05), as compared to non-treated (‘naïve’; Nv) and saline-treated (Sal) controls. The mean ± standard deviation CSD velocities of propagation (in mm/min) for the Sal, AA and Nv groups were respectively 3.75 ± 0.03, 4.26 ± 0.08 and 3.81 ± 0.04 for the W condition and 4.29 ± 0.08, 4.51 ± 0.04 and 4.30 ± 0.04 for the M groups. The results demonstrate a CSD-facilitation by AA regardless of nutritional status. They also suggest that, at the dose of 60 mg/kg/day chronically administered during brain development, AA may act as a prooxidant in brain, in view of the contrasting effect as compared with other antioxidants, which reduce CSD.     

Labisia pumila extract regulates body weight and adipokines in ovariectomized rats

Objective

We investigated the effects of a standardized water extract of Labisia pumila var. alata (LPva), and compared to estrogen replacement (ERT), on body weight gain, uterus weight, adipose tissue mRNA and protein levels of adipokines in ovariectomized (OVX) rats.

Methods

Eight-week-old OVX Sprague-Dawley rats were administered orally with either 10 mg/kg/day (LPva10), 20 mg/kg/day (LPva20) or 50 mg/kg/day (LPva50) of LPva for 30 days. Sham-operated (Sham) and estrogen-treated OVX rats (ERT, 0.625 mg/kg/day) served as controls. Plasma adipokines were measured, and mRNA expressions of the adipokines were determined in the adipose tissues.

Results

ERT- and LPva50-treated OVX rats showed significantly less (p < 0.05) weight gain compared to untreated OVX rats. Ovariectomy caused plasma leptin levels to decrease significantly (p < 0.05), but when treated with LPva or ERT, plasma leptin increased significantly to levels higher or comparable to that seen in the Sham group. The mRNA expression of leptin was higher in the LPva-treated animals than in all other groups. In contrast, the elevated plasma resistin concentrations in OVX rats were significantly reduced in rats given ERT (p < 0.05) and LPva extracts (p < 0.05). There was no difference in adiponectin levels in all groups. The uterus to body weight ratio of untreated OVX rats was significantly low compared to Sham (p < 0.05), but showed dose-dependent increase upon treatment with LPva.

Conclusion

The present study provides first evidence that LPva exerts uterotrophic effect and regulates body weight gain by modulating secretion of leptin and resistin, and expression of the adipokines in adipose tissues.     

A complex dietary supplement modulates nitrative stress in normal mice and in a new mouse model of nitrative stress and cognitive aging

We examined whether transgenic growth hormone mice (Tg) that exhibit accelerated cognitive aging and exceptional free radical damage also express elevated nitrative stress. We characterized age-related patterns of 3-nitrotyrosine (3-NT) in brain homogenate and mitochondria of Tg and normal (Nr) mice as modulated by a complex anti-aging dietary supplement. Levels of 3-NT rose rapidly with age in Tg brain homogenate whereas normal controls maintained constant lower levels. The age-related slope for 3-NT was 3.6-fold steeper in untreated Tg compared to treated Tg (p < 0.009), although treated Tg showed elevation in youth. Opposite to Tg, treated Nr mice had reduced 3-NT in youth (p < 0.02).The age-related pattern of mitochondrial 3-NT in Nr mice was parabolic (p < 0.005). Remarkably, levels in treated Nr were reduced by ∼50% (p < 0.0007). Untreated Tg showed strongly increasing mitochondrial 3-NT with higher mitochondrial activity (p < 0.01) whereas treated Tg showed lower nitrosylation at higher levels of mitochondrial activity. Tg mice also expressed a postural abnormality that is a biomarker of neurodegeneration and/or nitrative stress. Tg represent a promising new model of nitrative stress associated with brain deterioration and results provide proof of principle that complex dietary supplements may be ameliorating.     

Serotonergic receptor upregulation in cerebral cortex and down regulation in brainstem of streptozotocin induced diabetic rats: Antagonism by pyridoxine and insulin

Insulin secretion and glucose homeostasis is implicated through serotonergic function. Pyridoxine is involved in decarboxylation step in synthesis of serotonin. The present study was carried out to find the role of insulin in combination with pyridoxine on the concentrations of 5-HT and 5-HIAA, 5-HT receptor binding, 5-HTT gene expression and immunohistochemistry studies in the cerebral cortex and brainstem of streptozotocin induced diabetic rats. 5-HT content showed a significant decrease with a significant increase in 5-HIAA in cerebral cortex (p < 0.01) and brain stem (p < 0.001) in diabetic rats. 5-HT receptor binding parameters, B_max and K_d, showed a significant decrease (p < 0.001) in diabetic rats in cerebral cortex whereas in brainstem it showed a significant increase (p < 0.001) compared to control. Gene expression studies of 5-HTT in cerebral cortex showed a significant down regulation (p < 0.001) and in brainstem an upregulation (p < 0.001) in diabetic rats compared to control. Insulin and pyridoxine treatment to diabetic rats reversed the 5-HT content, B_max, K_d and gene expression of 5-HTT confirmed by immunohistochemistry studies in cerebral cortex and brainstem to near control. Thus our results suggest that pyridoxine along with insulin has a role in the regulation of insulin synthesis and release through serotonergic function which has clinical significance in the management of diabetes.     

Heme-a, the heme prosthetic group of cytochrome c oxidase,is increased in Alzheimer's disease

Heme-a, is the heme prosthetic group of cytochrome c oxidase (COX), the terminal complex of the mitochondrial electron transport chain. We measured heme-a levels in postmortem brain tissue from nine patients diagnosed with dementia: Alzheimer's disease (AD) was the primary diagnosis in five, AD/diffuse Lewy body disease (DLBD) was diagnosed in two, DLBD was diagnosed in one, and DLBD (severe)/AD (mild) was diagnosed in one. Eight non-demented patients who died from non-neurological causes served as controls. When the primary diagnosis was AD (AD and AD/DLBD), levels of cerebral heme-a were increased almost two-fold on a protein basis compared to controls (p < 0.001). Using perfused and non-perfused rats we showed that measured levels of cerebral heme-a were unaffected by the presence of blood in brain tissue. In mice we showed that levels of cerebral heme-a were unaffected by 24 h of storage at 4 °C prior to freezing. These animal studies suggest that increased levels of cerebral heme-a in AD were not due to blood in postmortem brain or variation in postmortem interval.