Zlatko Trkanjec

Nano-HPLC–MS analysis of phospholipids in cerebrospinal fluid of Alzheimer’s disease patients—a pilot study

There is emerging evidence that lipids play an important role in many neurodegenerative processes, for example in Alzheimer’s disease (AD). Although different lipid alterations in the AD brain have been reported, there have only been very few investigations of lipid changes in the cerebrospinal fluid (CSF). Recent developments in mass spectrometry (MS) have enabled fast and sensitive detection of lipid species in different biological matrixes. In this study we developed an on-line HPLC–MS method for phospholipid profiling in the CSF based on nano-HPLC separation using an Amide column and detection with electrospray (ESI) quadrupole–time of flight (QTOF) MS. We achieved good separation, reproducibility, and sensitivity in monitoring of the major phospholipid classes, phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylinositol (PI), and sphingomyelin (SM) in CSF. To emphasize the applicability of the method, a pilot study was performed on a group of CSF samples (N = 16) from individuals with probable AD and non-demented controls. We observed a statistically significant increase of SM levels (24.3 ± 2.4%) in CSF from probable AD individuals vs. controls. Our findings indicate that SM levels in the CSF could potentially provide a new lead in AD biomarker research, and show the potential of the method for disease-associated CSF phospholipid screening.

Brainstem raphe lesion in patients with major depressive disorder and in patients with suicidal ideation recorded on transcranial sonography

Recent transcranial sonography (TCS) studies showed that disruption of echogenic midbrain line, corresponding to basal limbic system and raphe nuclei (RN) within, might represent functional marker for the development of depression. Major depressive disorder (MDD) is one of the most common psychiatric disorders associated with suicidal ideation. We initiated this study to assess the usefulness of TCS recording in a group of MDD patients and in MDD patients who also reported suicidal ideation, on the assumption that TCS might serve as a screening method for differentiating patients at risk of suicide. Altogether 71 subjects: 17 patients with MDD, 14 patients with MDD who also reported suicidal ideation and 40 healthy controls, were studied using TCS by two independent physicians. Reduced raphe echogenicity was found in 8 of 17 (47%) of the patients with MDD but only in 6 of 40 (15%) controls. In patients with suicidal ideations that finding was even more pronounced (12 of 14, 86%) with the highest frequency of completely not visible TCS RN finding (10 of 14, 72%). Data showed that altered echogenicity of the RN is frequent in patients with suicidal ideation. Normal RN echogenicity in MDD patients was associated with less severe depressive symptoms and rarely with the presence of suicidal ideations. As far as we know, these are the first ever obtained results which show that TCS might help differentiating MDD patients with suicidal risk or eventually predict good disease recovery based on the findings of RN hypo- or normoechogenicity.Recent transcranial sonography (TCS) studies showed that disruption of echogenic midbrain line, corresponding to basal limbic system and raphe nuclei (RN) within, might represent functional marker for the development of depression. Major depressive disorder (MDD) is one of the most common psychiatric disorders associated with suicidal ideation. We initiated this study to assess the usefulness of TCS recording in a group of MDD patients and in MDD patients who also reported suicidal ideation, on the assumption that TCS might serve as a screening method for differentiating patients at risk of suicide. Altogether 71 subjects: 17 patients with MDD, 14 patients with MDD who also reported suicidal ideation and 40 healthy controls, were studied using TCS by two independent physicians. Reduced raphe echogenicity was found in 8 of 17 (47%) of the patients with MDD but only in 6 of 40 (15%) controls. In patients with suicidal ideations that finding was even more pronounced (12 of 14, 86%) with the highest frequency of completely not visible TCS RN finding (10 of 14, 72%). Data showed that altered echogenicity of the RN is frequent in patients with suicidal ideation. Normal RN echogenicity in MDD patients was associated with less severe depressive symptoms and rarely with the presence of suicidal ideations. As far as we know, these are the first ever obtained results which show that TCS might help differentiating MDD patients with suicidal risk or eventually predict good disease recovery based on the findings of RN hypo- or normoechogenicity.

Can beta stiffness index be proposed as risk factor for dementia

BACKGROUND AND AIM Changes of arterial stiffness indicate alteration in arterial mechanics predisposing to the evolution of stroke or vascular dementia. The aim of this pilot study was to investigate whether ultrasound parameters, particularly beta stiffness index (BSI), should be further explored as independent markers or risk factors for dementia. PATIENTS AND METHODS There were 38 demented patients included in this study (72.53+/-7.87 yrs) and 33 clinically healthy controls (68.85+/-3.52 yrs). Risk factors were noted and ultrasound measurements performed on common carotid artery (CCA) using eTracking software on Aloka ProSound ALPHA 10 with 13 MHz linear probe. Level of significance was p<0.05. RESULTS Arterial hypertension was present in 24 patients, atrial fibrillation in 9, diabetes in 3, and hypercholesterolemia in 18. Hypertension was present as a single risk factor in 15 controls with average diastolic blood pressure significantly lower in patients. Significantly higher in patients were mean intima-media thickness, systolic and diastolic CCA diameters (CD), and mean BSI in CCA bilaterally. Linear regression analysis for groups of Alzheimers dementia and vascular dementia proved that MMSE of the two groups relates to CCA diameter change (CDc) and BSI change explaining up to 5% of variability. CONCLUSIONS CD, CDc and BSI should be monitored in patients with cognitive decline and further explored as possible independent markers or risk factors; in future studies groups of demented and non-demented patients should be age, sex and risk factor matched.

Hemispheric asymmetries in blood flow during color stimulation

Visual stimulation influences mean blood flow velocities (MBFV) in posterior cerebral arteries (PCA). In 51 healthy, right-handed volunteers MBFV were measured in PCA with opened and closed eyes and while watching colored light (red, yellow, green, blue) for 1 minute. Volunteers had eyes closed 2 minutes between different colors. MBFV in left PCA was 41.2 ± 8.6 cm/s (mean ± 2SD) and 27.8 ± 8.5 cm/s with eyes opened and closed, respectively. For red light MBFV in left PCA was 31.4 ± 7.1 cm/s, for yellow 31.4 ± 7.2 cm/s, for green 32.0 ± 8.3 cm/s, and for blue 33.0 ± 7.6 cm/s. MBFV in right PCA 41.7 ± 8.9 cm/s and 28.2 ± 9.1 cm/s with eyes opened and closed, respectively. For red light MBFV in right PCA was 39.4 ± 8.4 cm/s, for yellow 38.9 ± 9.2 cm/s, for green 37.8 ± 9.4 cm/s and for blue 38.0 ± 8.8 cm/s. There was no significant difference in MBFV between left and right PCA with eyes opened and closed, but MBFV were significantly higher in right PCA for each color than corresponding MBFV in left PCA. These findings could indicate the greater metabolism of visual cortex in right occipital lobe while watching colors. Visual cortex of right occipital lobe could have greater importance in color perception than visual cortex of left occipital lobe.

Differences of blink-reflex abnormalities in patients suffering from idiopathic and symptomatic trigeminal neuralgia

SummaryWe investigated the brainstem blink reflex in patients suffering from idiopathic and symptomatic trigeminal neuralgia to establish possible dysfunction in the reflex cycle and determine eventual differences between the two disease types. The study included 35 patients with idiopathic disease and seven patients with symptomatic disease, their results compared with those of 50 normal controls. We stimulated the forehead afferents of the supraorbital nerve and recorded the response from both orbicularis oculi muscles. We tested latencies of bilateral late components (R2, R2′), irritative R3 component and the incidence of R3 component. The patients with idiopathic trigeminal neuralgia showed normal parameters of blink reflex, except for the greater incidence of R3 component. Patients with symptomatic trigeminal neuralgia showed prolonged latencies of R2, R2′ and R3 components when stimulating the afflicted side, but no significant difference in incidence of R3 component compared with the control group. The results indicate that abnormalities of blink reflex are significantly different in the two groups of patients. The high incidence of R3 component seems to be typical of idiopathic disease, whereas the prolonged latencies of late reflex components after stimulation of the afflicted side seem to be typical for symptomatic disease. These results suggest that testing the blink reflex may prove a significant aid in distinguishing the idiopathic and symptomatic disease types.ZusammenfassungWir untersuchten den Blinkreflex bei Patienten, die an einer idiopathischen bzw. einer symptomatischen Trigeminusneuralgie litten, um mögliche Funktionsstörungen im Reflexzyklus zu erfassen und um einen eventuellen Unterschied zwischen den Gruppen zu erheben. Untersucht wurden 35 Patienten mit idiopathischer und 7 Patienten mit symptomatischer Trigeminusneuralgie, verglichen mit einer Gruppe von 50 Kontrollpersonen. Nach elektrischer Stimulation an Foramen supraorbitale wurde die Reizantwort an beiden Mm. orbicularis oculi abgeleitet. Dabei wurden die Latenz der bilateralen späten Komponenten (R2, R2′) und der irritativen R3-Komponente sowie die Inzidenz der R3-Komponente gemessen. Die Patienten mit idiopathischer Trigeminusneuralgie zeigten bis auf eine höhere Inzidenz der R3-Komponente normale Parameter des Blinkreflexes. Die Patienten mit symptomatischer Trigeminusneuralgie wiesen im Vergleich zur Kontrollgruppe verlängerte Latenzen der R2-, R2′- und R3-Komponenten auf. Die Inzidenz der R3-Komponente zeigte keinen Unterschied zu Kontrollgruppe. Wir schließen aus unseren Ergebnissen, dass der Blinkreflex bei Trigeminusneuralgie je nach Erscheinungsform unterschiedlich beeinträchtigt ist. Eine hohe Inzidenz von R3-Komponenten scheint typisch für die idiopathische Form zu sein. Verlängerte Latenzzeiten der späten Reflexkomponenten nach Stimulation scheinen dahingegen typisch für die symptomatische Form der Erkrankung. Die Untersuchung des Blinkreflexes könnte daher zusätzliche Information für die Unterscheidung zwischen symptomatischer und idiopathischer Trigeminusneuralgie bieten.

Frequency and characteristics of emotional disorders in patients after ischemic stroke

Emotional disturbances in stroke patients may unfavorably affect the process of rehabilitation and longterm outcome of the disease. The aim of the study was to assess the prevalence of emotional disturbances and their characteristics in our stroke patients, according to hemispheric lateralization of cerebral lesion (as recorded by CT), patient sex and grade of neurological handicap (as assessed by Rankin scale). The study included 50 patients (29 men and 21 women, mean age 65.52 p ; 7.07 and 64.62 p ; 11.83 years, respectively) who had suffered ischemic stroke 3 weeks to 6 months before the study. The Crown-Crisp experience index which consists of six scales: scales of anxiety, phobia, obsession, somatization, depression and hysteria, were used for detection of emotional disturbances. Results showed a high prevalence of emotional disturbances in the study group. Depression was most common (36 of study patients), followed by generalized anxiety (n=29) and phobic disturbances (n=33). According to hemispheric lateralization of the cerebral lesion, a more intense emotional response was found in case of right hemispheric lesions, however, the difference was statistically significant only on the scale of somaticized anxiety (p<0.05). According to sex, a more intense emotional response was recorded in women. The difference being statistically significant on the scales of anxiety (p<0.05), depression (p<0.05) and phobia (p<0.01). An increasing tendency in the prevalence of emotional disturbances was observed with the increasing severity of neurologic deficit (p<0.05). Study results showed a high prevalence of emotional disturbances after ischemic stroke, among which the most common is depression.

Efficacy and safety of Ginkgo biloba standardized extract in the treatment of vascular cognitive impairment: a randomized, double-blind, placebo-controlled clinical trial

Objectives The aim of this randomized, double-blind, placebo-controlled trial was to determine the efficacy and safety of Ginkgo biloba extract in patients diagnosed with vascular cognitive impairment (VCI). Methods A total of 90 patients (aged 67.1±8.0 years; 59 women) were randomly allocated (1:1:1) to receive G. biloba 120 mg, G. biloba 60 mg, or placebo during a 6-month period. Assessment was made for efficacy indicators, including neuropsychological tests scores (Sandoz Clinical Assessment Geriatric Scale, Folstein Mini-Mental State Examination, Mattis Dementia Rating Scale, and Clinical Global Impression) and transcranial Doppler ultrasound findings. Safety indicators included laboratory findings, reported adverse reactions, and clinical examination. Results At the end of 6-month study period, G. biloba 120 and 60 mg showed a statistically significant positive effect in comparison with placebo only on the Clinical Global Impression score (2.6±0.8 vs 3.1±0.7 vs 2.8±0.7, respectively; P=0.038). The Clinical Global Impression score showed a significant deterioration from the baseline values in the placebo group (−0.3±0.5; P=0.021) as opposed to G. biloba groups. No significant differences were found in the transcranial Doppler ultrasound findings. Adverse reactions were significantly more common and serious in the placebo group (16 subjects) than in either of the two G. biloba extract groups (eight and nine subjects, respectively), whereas laboratory findings and clinical examinations revealed no differences between the groups receiving G. biloba extract and placebo. Conclusion According to our results, G. biloba seemed to slow down the cognitive deterioration in patients with VCI, but the effect was shown in only one of the four neuropsychological tests administered. However, because of this mild effect in combination with a few adverse reactions, we cannot say that it is ineffective or unsafe either. Further studies are still needed to provide unambiguous evidence on the efficacy and safety of G. biloba extract.

Transcranial sonography in movement disorders

Background: So far, there is no reliable test that can clearly distinguish between various movement disorders. Therefore, the diagnosis is still based on the clinical examination. However, the misdiagnosis rate for the most common movement disorders – Parkinson’ s disease (PD) and Essential tremor (ET) – in the early stages is as high as 20– 30% for PD and in about one of three patients in ET.We initiated these studies to assess the possibility of Transcranial sonography (TCS) to help differentiate PD from ET by measuring echogenicity of the substantia nigra (SN). Patients and methods: Our study included 60 patients with PD, 30 patients with ET and 60 healthy controls. The TCS recordings were done in axial plane by standardized protocol by two independent investigators. SN was displayed, encircled, and measured two times. Mean area was calculated. Mann Whitney U test for intergroup comparison was applied. Results: Patients with ET and PD had mean SN size of 0.15 cm2 (± ; 0.04) and 0.27cm2 (± ; 0.06), which showed significant difference (p<0.001). In the control group bilateral combined mean SN size was 0.17cm2 (± ; 0.06), which was significantly different from PD group (p<0.001), but not from ET group (p=0.240). Conclusion: The measurement of SN by means of TCS is a valuable tool in the differentiation of movement disorders. Due to portability, lack of invasiveness and easiness of reproducibility, TCS might help in diagnosing PD or in differential diagnosis of doubtful clinical cases.

Feasibility of transcranial Doppler and single photon emission computed tomography in compound neuroactivation task

The aim of this study was to test feasibility of transcranial Doppler (TCD) and single photon emission computed tomography (SPECT) during compound neuroactivation task. The study was performed in 60 healthy right-handed volunteers. Cerebral blood flow velocity was measured by TCD in both middle cerebral arteries (MCA) at baseline and during computer game. The same stimulus and response pattern was used in 15 subjects that additionally underwent brain SPECT. Percentage differences between measurements were determined through quantitative result assessment. Both methods detected a statistically significant cerebral blood flow increase during neuroactivation. Correlation of TCD and SPECT showed statistically significant correlation only for the increase of cerebral blood flow velocity in the right MCA and for the right-sided cerebral blood flow increase, demonstrating that both methods partially measure similar cerebral blood flow changes that occur during neuroactivation. Comparison of TCD and SPECT showed TCD to be inadequately sensitive method for evaluation of cerebral blood flow during complex activation paradigm.

Non-invasive assessment of cerebral blood flow changes during complex activation

BackgroundThe aim of this study was to cross-validate functional transcranial Doppler (FTCD) and brain single photon emission computed tomography (SPECT) using complex activation paradigm and to test the feasibility of FTCD in complex neuroactivation research.MethodologyThe study was performed in the group of 60 healthy, right-handed subjects. Cerebral blood flow velocity (CBFV) was measured in both middle cerebral arteries (MCA) during baseline and during computer game interaction, using FTCD. Identical stimulus and response patterns were used in the subgroup of 15 subjects that underwent brain SPECT. Quantitative assessment of results was done to detect the percentage variation between the two measurements.ResultsA statistically significant increase of cerebral blood flow (CBF) was detected by FTCD and brain SPECT (p<0.0001). In comparison between the two methods, statistically significant association (r=0.5608, P=0.030, coefficient of determination r2 = 0.3154) was detected only for CBFV increase in the right MCA and for the rightsided cerebral blood perfusion (CBP) increase, indicating that these two methods only partially measure the same characteristics associated with activation of specific brain areas.ConclusionIn comparison with SPECT, FTCD is not sufficiently sensitive method for evaluation of CBF changes during complex activation paradigm. Our study represents negative evidence and stand against the common belief that FTCD is as good as other neuroimaging methods used for CBF measurements during neuroactivation. Therefore, FTCD might not be a reliable and suitable method for evaluation of CBF changes during complex neuroactivation paradigm.