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Abstract Background Structural analysis of biochemical networks is a growing field in bioinformatics and systems biology. The availability of an increasing amount of biological data from molecular biological networks promises a deeper understanding but confronts researchers with the problem of combinatorial explosion. The amount of qualitative network data is growing much faster than the amount of quantitative data, such as enzyme kinetics. In many cases it is even impossible to measure quantitative data because of limitations of experimental methods, or for ethical reasons. Thus, a huge amount of qualitative data, such as interaction data, is available, but it was not sufficiently used for modeling purposes, until now. New approaches have been developed, but the complexity of data often limits the application of many of the methods. Biochemical Petri nets make it possible to explore static and dynamic qualitative system properties. One Petri net approach is model validation based on the computation of the system's invariant properties, focusing on t-invariants. T-invariants correspond to subnetworks, which describe the basic system behavior. With increasing system complexity, the basic behavior can only be expressed by a huge number of t-invariants. According to our validation criteria for biochemical Petri nets, the necessary verification of the biological meaning, by interpreting each subnetwork (t-invariant) manually, is not possible anymore. Thus, an automated, biologically meaningful classification would be helpful in analyzing t-invariants, and supporting the understanding of the basic behavior of the considered biological system. Methods Here, we introduce a new approach to automatically classify t-invariants to cope with network complexity. We apply clustering techniques such as UPGMA, Complete Linkage, Single Linkage, and Neighbor Joining in combination with different distance measures to get biologically meaningful clusters (t-clusters), which can be interpreted as modules. To find the optimal number of t-clusters to consider for interpretation, the cluster validity measure, Silhouette Width, is applied. Results We considered two different case studies as examples: a small signal transduction pathway (pheromone response pathway in Saccharomyces cerevisiae) and a medium-sized gene regulatory network (gene regulation of Duchenne muscular dystrophy). We automatically classified the t-invariants into functionally distinct t-clusters, which could be interpreted biologically as functional modules in the network. We found differences in the suitability of the various distance measures as well as the clustering methods. In terms of a biologically meaningful classification of t-invariants, the best results are obtained using the Tanimoto distance measure. Considering clustering methods, the obtained results suggest that UPGMA and Complete Linkage are suitable for clustering t-invariants with respect to the biological interpretability. Conclusion We propose a new approach for the biological classification of Petri net t-invariants based on cluster analysis. Due to the biologically meaningful data reduction and structuring of network processes, large sets of t-invariants can be evaluated, allowing for model validation of qualitative biochemical Petri nets. This approach can also be applied to elementary mode analysis.

Background and Purpose: Sarcoidosis is a granulomatous disease of unknown etiology affecting the central nervous system in up to 15% of the patients. Diagnosis of neurosarcoidosis is very challenging due to the heterogeneity of its clinical manifestation. This study intended to evaluate the distribution of cerebral lesion sites and the potential presence of specific lesion clusters in neurosarcoidosis patients using voxel-based lesion symptom mapping (VLSM)., Methods: Patients with neurosarcoidosis were retrospectively identified and included between 2011 and 2022. Cerebral lesion sites were correlated voxel-wise with presence and absence of neurosarcoidosis using non-parametric permutation test. Multiple sclerosis patients served as controls for the VLSM-analysis., Results: Thirty-four patients (mean age 52 ± 15 years) of whom 13 were diagnosed with possible, 19 with probable and 2 with confirmed neurosarcoidosis were identified. Lesion overlap of neurosarcoidosis patients demonstrated a distribution of white matter lesions in all brain areas, with a periventricular predilection similar to multiple sclerosis. In contrast to multiple sclerosis controls, no propensity for lesions in proximity of the corpus callosum was observed. Neurosarcoidosis lesions appeared smaller and lesion volume was lower in the neurosarcoidosis cohort. The VLSM analysis showed minor associations between neurosarcoidosis and damaged voxels in the bilateral frontobasal cortex., Conclusions: The VLSM analysis yielded significant associations in the bilateral frontal cortex, suggesting that leptomeningeal inflammatory disease with following cortical involvement is a quite specific feature in neurosarcoidosis. Lesion load was lower in neurosarcoidosis than in multiple sclerosis. However, no specific pattern of subcortical white matter lesions in neurosarcoidosis was revealed., (© 2023. The Author(s).)

Introduction: Myocardial injury related to acute ischaemic stroke is common even without primary cardiac disease. We intended to determine associations between values of left ventricular ejection fraction (LVEF) and ischaemic stroke lesion sites., Methods: Of a local database, patients with acute first-ever ischaemic stroke confirmed by brain imaging but without pre-existing heart disease were included. The cardiac morphology and LVEF were obtained from transthoracic or transesophageal echocardiography, and impaired LVEF was categorised as mild (35%-50%), moderate (34%-25%) and severe (<25%). Patient age, stroke severity, ischaemic lesion volume, prevalence of troponin I increase (>0.1 ng/mL), atrial fibrillation and cardiac wall motion abnormalities were assessed and compared between patients with and without impaired LVEF after stroke (significance: p<0.05). A multivariate voxelwise lesion analysis correlated LVEF after stroke with sites of ischaemic lesions., Results: Of 1209 patients who had a stroke, 231 (mean age 66.3±14.0 years) met the inclusion criteria; 40 patients (17.3%) had an impaired LVEF after stroke. Patients with impaired LVEF had higher infarct volumes (53.8 mL vs 30.0 mL, p=0.042), a higher prevalence of troponin increase (17.5% vs 4.2%, p=0.006), cardiac wall motion abnormalities (42.5% vs 5.2%, p<0.001) and atrial fibrillation (60.0% vs 26.2%, p<0.001) than patients with LVEF of >50%. The multivariate voxelwise lesion analysis yielded associations between decreased LVEF and damaged voxels in the insula, amygdala and operculum of the right hemisphere., Conclusion: Our imaging analysis unveils a prominent role of the right hemispheric central autonomic network, especially of the insular cortex, in the brain-heart axis. Our results support preliminary evidence that acute ischaemic stroke in distinct brain regions of the central autonomic network may directly impair cardiac function and thus further supports the concept of a distinct stroke-heart syndrome., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background and Purpose: The relationship between ischemic stroke site and occurrence of poststroke epilepsy (PSE) is incompletely understood. This study intended to evaluate incidence and temporal profiles of seizures and to correlate ischemic lesion sites with PSE using voxel-based lesion symptom mapping (VLSM)., Methods: Patients with imaging-confirmed first-ever ischemic stroke without prior history of epilepsy were prospectively included. Demographic data, cardiovascular risk factors, and National Institute of Health Stroke Scale (NIHSS) scores were assessed. Data on seizures and modified Rankin scale scores were determined within a 90-day period after stroke onset. Ischemic lesion sites were correlated voxel wise with occurrence of PSE using nonparametric permutation test. Age- and sex-matched patients with first-ever ischemic strokes without PSE after 90 days served as controls for the VLSM analysis., Results: The stroke database contained 809 patients (mean age: 68.4 ± 14.2 years) with first-ever imaging-confirmed ischemic strokes without history of epilep. Incidence of PSE after 90-day follow-up was 2.8%. Five additional patients were admitted to the emergency department with a seizure after 90-day follow-up. Fifty percent of the seizures occurred in the acute phase after stroke. PSE patients had higher NIHSS scores and infarct volumes compared to controls without PSE (p < .05). PSE patients had infarcts predominantly involving the cerebral cortex. The hemisphere-specific VLSM analysis shows associations between PSE and damaged voxels in the left-hemispheric temporo-occipital transition zone., Conclusions: The data indicate that PSE occurs in a small proportion of patients with rather large ischemic strokes predominantly involving the cerebral cortex. Especially patients with ischemic lesions in the temporo-occipital cortex are vulnerable to develop PSE., (© 2023 The Authors. Journal of Neuroimaging published by Wiley Periodicals LLC on behalf of American Society of Neuroimaging.)

Purpose: Financial toxicity is a well-recognized problem for patients with cancer. However, a crucial gap remains in describing and implementing mitigation strategies. We conducted a national survey of a multiethnic adolescent/pediatric and adult patient population served by Family Reach, a nonprofit organization focused on removing financial barriers to cancer care, to evaluate the impact of a comprehensive financial resource on patient-reported financial toxicity., Methods: An electronic survey was administered to characterize patients' current financial health and the impact of Family Reach's resources on financial toxicity. The survey was e-mailed to all patients or caregivers who received resources from Family Reach between January 1, 2020, and June 30, 2020. Factors associated with higher financial stress and higher potential impact of resources on financial burden were evaluated through separate multivariate regression models. Qualitative responses were analyzed using manual coding and thematic analysis., Results: Three hundred thirty socioeconomically and racially diverse respondents (overall response rate 40%; 46% non-Hispanic White; 48% with incomes below the federal poverty line) completed the survey and were included in the analysis. More than half of respondents reported high financial stress in the previous week. Hispanic ethnicity, Black race, and low annual household income were associated with higher financial toxicity. A greater amount of financial assistance was associated with a higher confidence rating that resources provided would decrease financial stress. In open-ended comments, respondents highlighted the impact of the COVID-19 pandemic and resulting job loss on financial toxicity, the importance of financial navigation, the benefits of financial assistance, and anxiety about long-term financial health., Conclusion: A comprehensive financial resource, particularly financial assistance, alleviated financial toxicity in a multiethnic national sample of patients with cancer. Ongoing work is critical to address sustainable funding sources and financial navigation to support patients during treatment and survivorship.

Objective: Restless legs syndrome (RLS) is known to be associated with multiple sclerosis (MS) and may be caused by MS lesions in specific cerebral brain regions. Applying a voxel-wise lesion analysis, we tried to identify the contribution of cerebral MS lesions to RLS., Methods: In this retrospective study, we established a cohort of people with MS with documented RLS and controls of people with MS without RLS matched disease severity. Diagnosis of MS and RLS was based on the current guidelines. The MS lesions were analyzed on T2-weighted magnetic resonance imaging scans (1.5 or 3 T). After manual delineation, lesion maps were converted into stereotaxic space. We generated a lesion overlap and performed a Liebermeister test with 4000 permutations to compare the absence or presence of RLS voxel-wise between patients with and without lesions in a given voxel., Results: Forty of the patients with RLS and MS fulfilled the inclusion criteria. The voxel-wise analysis yielded associations between RLS and MS in the subcortex of the left gyrus precentralis., Conclusion: Our voxel-wise analysis shows associations in the subcortex of the left gyrus precentralis. Thus, our data suggests that a dysfunction of the efferent motor system due to cerebral lesions may contribute to the pathophysiology of RLS in MS., (© 2022. The Author(s).)

Objective: To determine patients' characteristics and regions in the temporal lobe where resections lead to a decline in picture naming., Methods: 311 patients with left hemispheric dominance for language were included who underwent epilepsy surgery at the Epilepsy Center of Erlangen and whose picture naming scores (Boston Naming Test, BNT) were available preoperatively and 6-months postoperatively. Surgical lesions were mapped to an averaged template based on preoperative and postoperative MRI using voxel-based lesion-symptom mapping (VBLSM). Postoperative brain shifts were corrected. The relationship between lesioned brain areas and the presence of a postoperative naming decline was examined voxel-wise while controlling for effects of overall lesion size at first in the total cohort and then restricted to temporal lobe resections., Results: In VBLSM in the total sample, a decline in BNT score was significantly related to left temporal surgery. When only considering patients with left temporal lobe resections (n = 121), 40 (33.1%) significantly worsened in BNT postoperatively. VBLSM including all patients with left temporal resections generated no significant results within the temporal lobe. However, naming decline of patients with epilepsy onset after 5 years of age was significantly associated with resections in the left inferior temporal (extent of BNT decline range: 10.8- 14.4%) and fusiform gyrus (decline range: 12.1-18.4%)., Significance: Resections in the posterior part of the dominant fusiform and inferior temporal gyrus was associated with a risk of deterioration in naming performance at six months after surgery in patients with epilepsy onset after 5 years of age but not with earlier epilepsy onset., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: Endovascular therapy (EVT) of large-vessel occlusion in acute ischemic stroke (AIS) may be performed in general anesthesia (GA) or conscious sedation (CS). We intended to determine the contribution of ischemic cerebral lesion sites on the physician's decision between GA and CS using voxel-based lesion symptom mapping (VLSM)., Methods: In a prospective local database, we sought patients with documented AIS and EVT. Age, stroke severity, lesion volume, vigilance, and aphasia scores were compared between EVT patients with GA and CS. The ischemic lesions were analyzed on CT or MRI scans and transformed into stereotaxic space. We determined the lesion overlap and assessed whether GA or CS is associated with specific cerebral lesion sites using the voxel-wise Liebermeister test., Results: One hundred seventy-nine patients with AIS and EVT were included in the analysis. The VLSM analysis yielded associations between GA and ischemic lesions in the left hemispheric middle cerebral artery territory and posterior circulation areas. Stroke severity and lesion volume were significantly higher in the GA group. The prevalence of aphasia and aphasia severity was significantly higher and parameters of vigilance lower in the GA group., Conclusions: The VLSM analysis showed associations between GA and ischemic lesions in the left hemispheric middle cerebral artery territory and posterior circulation areas including the thalamus that are known to cause neurologic deficits, such as aphasia or compromised vigilance, in AIS-patients with EVT. Our data suggest that higher disability, clinical impairment due to neurological deficits like aphasia, or reduced alertness of affected patients may influence the physician's decision on using GA in EVT., (© 2021. The Author(s).)

Background and Purpose: Initial cardiovascular fingolimod effects might compromise baroreflex responses to rapid blood pressure (BP) changes during common Valsalva-like maneuvers. This study evaluated cardiovascular responses to Valsalva maneuver (VM)-induced baroreceptor unloading and loading upon fingolimod initiation., Patients and Methods: Twenty-one patients with relapsing-remitting multiple sclerosis performed VMs before and 0.5, 1, 2, 3, 4, 5, and 6 hours after fingolimod initiation. We recorded heart rate (HR) as RR intervals (RRI), systolic and diastolic BP (BPsys, BPdia) during VM phase 1, VM phase 2 early, VM phase 2 late, and VM phase 4. Using linear regression analysis between decreasing BPsys and RRI values during VM phase 2 early, we determined baroreflex gain (BRG) reflecting vagal withdrawal and sympathetic activation upon baroreceptor unloading. To assess cardiovagal activation upon baroreceptor loading, we calculated Valsalva ratios (VR) between maximal and minimal RRIs after strain release. Analysis of variance or Friedman tests with post hoc analysis compared corresponding parameters at the eight time points (significance: p < 0.05)., Results: RRIs at VM phase 1, VM phase 2 early, and VM phase 2 late were higher after than before fingolimod initiation, and maximal after 4 hours. Fingolimod did not affect the longest RRIs upon strain release, but after 3, 5, and 6 hours lowered the highest BPsys values during overshoot and all BPdia values, and thus reduced VRs. BRG was slightly higher after 3 and 5 hours, and significantly higher after 4 hours than before fingolimod initiation., Conclusions: VR-decreases 3-6 hours after fingolimod initiation are physiologic results of fingolimod-associated attenuations of BP and HR increases at the end of strain and do not suggest impaired cardiovagal activation upon baroreceptor loading. Stable and at the time of HR nadir significantly increased BRGs indicate improved responses to baroreceptor unloading. Thus, cardiovascular fingolimod effects do not impair autonomic responses to sudden baroreceptor loading or unloading but seem to be mitigated by baroreflex resetting.

Background: In a previous study, we reported that selective dorsal root ganglion stimulation (DRG STIM ) at DRG level L4 promoted a favorable outcome for complex regional pain syndrome (CRPS) patients along with DRG STIM -related changes of inflammatory biomarkers in blood and saliva. The impact on somatosensation is largely unknown. Herein, we assessed the quantitative sensory profile to quantify L4-DRG STIM effects in CRPS patients., Methods: Twelve refractory CRPS patients (4 female; 8 male; mean age 69 ± 9 years) received standardized quantitative sensory testing (QST) protocol at baseline and after 3 months of unilateral L4-DRG STIM assessing nociceptive and non-nociceptive thermal and mechanical sensitivity of the knee affected by CRPS and the contralateral non-painful knee area., Results: At baseline, CRPS subjects showed significantly increased thresholds for warmth, tactile and vibration detection (WDT, MDT and VDT) and exaggerated pain summation (WUR). After 3 months of unilateral L4-DRG STIM all pain parameters exhibited trends towards normalization of sensitivity accumulating to a significant overall normalization for pain sensitivity (effect size: 0.91, p < 0.01), while with the one exception of WDT all non-nociceptive QST parameters remained unchanged. Overall change of non-nociceptive detection was negligible (effect size: 0.25, p > 0.40). Notably, reduction of pain summation (WUR) correlated significantly with pain reduction after 3 months of L4-DRG STIM ., Conclusions: Selective L4-DRG STIM lowered ongoing pain in CRPS patients and evoked significant normalization in the pain domain of the somatosensory profile. Thermoreception and mechanoreception remained unchanged. However, larger randomized, sham-controlled trials are highly warranted to shed more light on effects and mechanisms of dorsal root ganglion stimulation on quantitative sensory characteristics. The study protocol was registered at the 15.11.2016 on German Register for Clinical Trials (DRKS ID 00011267). https://www.drks.de/drks&#95;web/navigate.do?navigationId=trial.HTML&TRIAL&#95;ID=DRKS00011267.

Introduction: Noninvasive temperature modulation by localized neck cooling might be desirable in the prehospital phase of acute hypoxic brain injuries. While combined head and neck cooling induces significant discomfort, peripheral vasoconstriction, and blood pressure increase, localized neck cooling more selectively targets blood vessels that supply the brain, spares thermal receptors of the face and skull, and might therefore cause less discomfort cardiovascular side effects compared to head- and neck cooling. The purpose of this study is to assess the effects of noninvasive selective neck cooling on cardiovascular parameters and cerebral blood flow velocity (CBFV)., Methods: Eleven healthy persons (6 women, mean age 42 ± 11 years) underwent 90 min of localized dorsal and frontal neck cooling (EMCOOLS Brain.Pad™) without sedation. Before and after cooling onset, and after every 10 min of cooling, we determined rectal, tympanic, and neck skin temperatures. Before and after cooling onset, after 60- and 90-min cooling, we monitored RR intervals (RRI), systolic, diastolic blood pressures (BPsys, BPdia), laser Doppler skin blood flow (SBF) at the index finger pulp, and CBFV at the proximal middle cerebral artery (MCA). We compared values before and during cooling by analysis of variance for repeated measurements with post hoc analysis (significance: p < 0.05)., Results: Neck skin temperature dropped significantly by 9.2 ± 4.5 °C (minimum after 40 min), while tympanic temperature decreased by only 0.8 ± 0.4 °C (minimum after 50 min), and rectal temperature by only 0.2 ± 0.3 °C (minimum after 60 min of cooling). Index finger SBF decreased (by 83.4 ± 126.0 PU), BPsys and BPdia increased (by 11.2 ± 13.1 mmHg and 8.0 ± 10.1 mmHg), and heart rate slowed significantly while MCA-CBFV remained unchanged during cooling., Conclusions: While localized neck cooling prominently lowered neck skin temperature, it had little effect on tympanic temperature but significantly increased BP which may have detrimental effects in patients with acute brain injuries.

Introduction: MR-guided focused ultrasound operating at higher intensities have been reported to effectively and precisely ablate deeper brain structures like the basal ganglia or the thalamic nuclei for the treatment of refractory movement disorders, neuropathic pain and most recently neuropsychiatric disorders, while low-intensity focused ultrasound represents an approach promoting mechanical blood-brain-barrier opening and neuromodulation. This narrative review summarizes the technical development and the therapeutic potential of incisionless MRgFUS in order to treat neuropsychiatric disorders., Areas Covered: A narrative review of clinical trials assessing the safety and efficacy of MRgFUS. A literature review was performed using the following search terms: MR-guided focused ultrasound, psychiatric disorders, noninvasive and invasive brain modulation/stimulation techniques., Expert Opinion: MRgFUS ablation is under clinical investigation (unblinded study design) for obsessive-compulsive disorders (OCDs) [capsulotomy; ALIC] and depression/anxiety disorders [capsulotomy] and has demonstrated an improvement in OCD and depression, although of preliminary character. Low-intensity ultrasound applications have been explored in Alzheimer´s disease (phase 1 study) and healthy subjects. Currently, limited evidence hinders comparison and selection between MRgFUS and noninvasive/invasive brain modulation therapies. However, comparative, sham-controlled trials are needed to reexamine the preliminary findings for the treatment of psychiatric disorders.

Aims: Besides spinal lesions, urinary incontinence may be attributed to particular cerebral lesion sites in multiple sclerosis (MS) patients. We intended to determine the contribution of suprapontine lesions to urinary incontinence in MS using a voxel-wise lesion analysis., Methods: In this retrospective study, we sought MS patients with documented urinary incontinence in a local database. We established a control group of MS-patients without documented urinary incontinence matched for gender, age, and disease severity. Patients with urinary incontinence due to local diseases of the urinary tract were excluded. The MS lesions were analyzed on T2-weighted magnetic resonance imaging scans (1.5 or 3T). After manual delineation and transformation into stereotaxic space, we determined the lesion overlap and compared the presence or absence of urinary incontinence voxel-wise between patients with and without lesions in a given voxel performing the Liebermeister test with 4000 permutations., Results: A total of 56 patients with urinary incontinence and MS fulfilled the criteria and were included. The analysis yielded associations between urinary incontinence and MS in the frontal white matter, temporo-occipital, and parahippocampal regions., Conclusions: Our voxel-wise analysis indicated associations between self-reported urinary incontinence and lesions in the left frontal white matter and right parahippocampal region. Thus, our data suggest that dysfunction of supraspinal bladder control due to cerebral lesions may contribute to the pathophysiology of urinary incontinence in MS., (© 2019 Wiley Periodicals, Inc.)

Patients with a history of mild traumatic brain injury (post-mTBI patients) may have enduring cardiovascular-autonomic dysregulation and emotional problems. Olfactory stimulation (OS) triggers emotional and cardiovascular-autonomic responses that might be compromised in post-mTBI patients. We therefore evaluated these responses to OS in post-mTBI patients. In 17 post-mTBI patients (interval since mTBI: 32.4 ± 6.8 months) and 17 age- and sex-matched controls, we recorded respiration, electrocardiographic RR intervals, and systolic and diastolic blood pressures (BPsys, BPdia) before and during pleasant vanilla stimulation and unpleasant hydrogen sulphide (H2S) stimulation. Participants rated OS-related pleasantness, arousal, intensity, and familiarity on 9-point Likert scales. Analyses of variance (ANOVAs) with post hoc analyses compared parameters within each group before and during OS. To assess associations between pleasantness, arousal, intensity, and familiarity, we correlated OS scores within groups (significance: p  < 0.05). Baseline parameters were similar between groups. Only in controls, vanilla stimulation significantly lowered BPsys and BPdia, whereas H2S stimulation lowered RR intervals. Vanilla-related pleasantness scores were lower, intensity scores were higher in patients than controls. During vanilla stimulation, pleasantness scores correlated negatively with arousal scores in controls, whereas familiarity scores correlated positively with intensity scores in patients. During H2S stimulation, familiarity scores correlated negatively with pleasantness scores in controls, whereas pleasantness scores correlated negatively with arousal scores in mTBI patients. Post-mTBI patients could not change BP or RR intervals during OS but perceived vanilla stimuli as less pleasant and more intense than did controls. Associations between pleasantness, arousal, intensity, and familiarity differed between groups suggesting different activation of the olfactory network and the central autonomic network upon OS. Subtle lesions within these networks might cause persistent changes in emotional and cognitive odor perception and cardiovascular responses.

Cardiovascular autonomic dysfunction is common in multiple sclerosis (MS) and contributes significantly to disability. We hypothesized that cerebral MS-lesions in specific areas of the central autonomic network might account for imbalance of the sympathetic and parasympathetic cardiovascular modulation. Therefore, we used voxel-based lesion symptom mapping (VLSM) to determine associations between cardiovascular autonomic dysfunction and cerebral MS-related lesion sites. In 74 MS-patients (mean age 37.0 ± 10.5 years), we recorded electrocardiographic RR-intervals and systolic and diastolic blood pressure. Using trigonometric regressive spectral analysis, we assessed low (0.04-0.15 Hz) and high (0.15-0.5 Hz) frequency RR-interval-and blood pressure-oscillations and determined parasympathetically mediated RR-interval-high-frequency modulation, mainly sympathetically mediated RR-interval-low-frequency modulation, sympathetically mediated blood pressure-low-frequency modulation, and the ratios of sympathetic and parasympathetic RR-interval-modulation as an index of sympathetic-parasympathetic balance. Cerebral MS-lesions were analyzed on imaging scans. We performed a VLSM-analysis correlating parameters of autonomic dysfunction with cerebral MS-lesion sites. The VLSM-analysis showed associations between increased RR-interval low-frequency/high-frequency ratios and lesions most prominently in the left insular, hippocampal, and right frontal inferior opercular region, and a smaller lesion cluster in the right middle cerebellar peduncle. Increased blood pressure-low-frequency powers were associated with lesions primarily in the right posterior parietal white matter and again left insular region. Our data indicate associations between a shift of cardiovascular sympathetic-parasympathetic balance toward increased sympathetic modulation and left insular and hippocampal lesions, areas of the central autonomic network. The VLSM-analysis further distinguished between right inferior fronto-opercular lesions disinhibiting cardiac sympathetic activation and right posterior parietal lesions increasing sympathetic blood pressure modulation., (© 2019 The Authors. Human Brain Mapping published by Wiley Periodicals, Inc.)

Background and Purpose- Oral angioedema (OA) is a rare but life-threatening complication in patients with ischemic stroke receiving intravenous thrombolysis with r-tPA (recombinant tissue-type plasminogen activator). This study intended to determine associations between thrombolysis-related OA and ischemic stroke lesion sites using a voxel-wise lesion analysis. Methods- Prospective registry data were used to identify ischemic stroke patients with thrombolysis-related OA between 2002 and 2018. For the study registry, ethics approval was obtained by the Ethics Committee of the Friedrich-Alexander Universität (FAU) Erlangen-Nürnberg (clinical registry registration: 377&#95;17Bc). Ischemic stroke patients with thrombolysis treatment but without OA admitted in the years 2011 and 2012 comprised the control group. Ischemic lesions were manually outlined on magnetic resonance imaging (1.5T or 3T) or computed tomographic scans and transformed into stereotaxic space. We determined the lesion overlap and compared the absence or presence of OA voxel-wise between patients with and without lesions in a given voxel using the Liebermeister test. Stroke severity was rated using the National Institutes of Health Stroke Scale score, and blood pressure, heart rate, blood glucose levels, and body temperature were determined on admission. Results- Fifteen ischemic stroke patients with thrombolysis-related OA were identified. The voxel-wise analysis yielded associations between OA and ischemic lesions in the insulo-opercular region with a right hemispheric dominance. Mean blood pressure was significantly lower in patients with OA than in controls. Age, National Institutes of Health Stroke Scale scores, infarct volumes, heart rate, and blood glucose levels did not differ between patients with and without OA. Conclusions- The voxel-wise analysis linked thrombolysis-related OA to right insulo-opercular lesions. The lower blood pressure in patients with thrombolysis-related OA may reflect bradykinin effects causing vasodilatation and increasing vascular permeability.

Background and Purpose: Bowel incontinence in multiple sclerosis might be associated with specific lesion sites. This study intended to determine associations between bowel incontinence and cerebral multiple sclerosis lesions using a voxel-wise lesion symptom mapping analysis., Methods: We conducted a retrospective study of multiple sclerosis patients with self-reported bowel incontinence and matched controls. Lesions were manually outlined on T2-weighted MRI scans and transformed into stereotaxic space. We performed a voxel-wise subtraction analysis subtracting the lesion overlap of patients without from patients with bowel incontinence. Finally, we compared the absence or presence of bowel incontinence between patients with and without lesions in a given voxel using the Liebermeister test., Results: A total of 51 patients were included in the study. The analysis yielded associations between bowel incontinence and lesions in the supramarginal gyrus of the left secondary somatosensory cortex and another lesion cluster in the right parahippocampal gyrus and amygdala., Conclusions: Our analysis indicates associations between bowel incontinence and lesions in the left supramarginal gyral area contributing to integrating anorectal-visceral sensation and in the right parahippocampal gyrus and amygdala contributing to generating visceral autonomic arousal states. Moreover, our results suggest left hemispheric dominance of sensory visceral integration, while limbic areas of the right hemisphere seem to contribute to the autonomic component of the defecation process. A limitation of our study is the retrospective evaluation of the bowel incontinence status based on medical records. Further research should evaluate the bowel incontinence status in multiple sclerosis patients prospectively to overcome the limitations of the current study., (© 2018 by the American Society of Neuroimaging.)

Secondary paroxysmal dyskinesia is a rare but life-quality-compromising symptom in multiple sclerosis (MS) and might be associated with particular MS lesions. The present study intended to determine associations between paroxysmal dyskinesia and the MS-associated lesion site using a voxelwise lesion analysis. We conducted a retrospective study and sought MS patients with documented paroxysmal dyskinesia and controls without paroxysmal dyskinesia matched for age, disease severity, and disease duration in a local database. The MS lesions were analysed on T2-weighted magnetic resonance imaging scans (1.5 or 3 T), manually outlined, and transformed into stereotaxic space. We determined the lesion overlap and compared the absence or presence of paroxysmal dyskinesia voxelwise between patients with and without lesions in a given voxel using the Liebermeister test with 4000 permutations. From 15,869 MS patient records screened, we identified 25 patients with paroxysmal dyskinesia. The voxelwise analysis in 22 subjects yielded associations between paroxysmal dyskinesia and MS lesions in the internal capsule, the basal ganglia, and another prominent lesion cluster in the posterior periventricular white matter. Our voxelwise analysis shows associations between paroxysmal dyskinesia and MS lesions in the internal capsule and basal ganglia, areas contributing to motor sequence programming. This association in another lesion site located in the posterior thalamic radiation suggests that lesions in subcortical sensory pathways may also contribute to paroxysmal dyskinesia in MS.

Background It has been proposed that multiple sclerosis lesions afflicting the pontine trigeminal afferents contribute to trigeminal neuralgia in multiple sclerosis. So far, there are no imaging studies that have evaluated interactions between supratentorial lesions and trigeminal neuralgia in multiple sclerosis patients. Methods We conducted a retrospective study and sought multiple sclerosis patients with trigeminal neuralgia and controls in a local database. Multiple sclerosis lesions were manually outlined and transformed into stereotaxic space. We determined the lesion overlap and performed a voxel-wise subtraction analysis. Secondly, we conducted a voxel-wise non-parametric analysis using the Liebermeister test. Results From 12,210 multiple sclerosis patient records screened, we identified 41 patients with trigeminal neuralgia. The voxel-wise subtraction analysis yielded associations between trigeminal neuralgia and multiple sclerosis lesions in the pontine trigeminal afferents, as well as larger supratentorial lesion clusters in the contralateral insula and hippocampus. The non-parametric statistical analysis using the Liebermeister test yielded similar areas to be associated with multiple sclerosis-related trigeminal neuralgia. Conclusions Our study confirms previous data on associations between multiple sclerosis-related trigeminal neuralgia and pontine lesions, and showed for the first time an association with lesions in the insular region, a region involved in pain processing and endogenous pain modulation.

Erectile function (EF) is frequently compromised in men with multiple sclerosis (MS). Functional neuroimaging in healthy men identified a network of brain areas, such as the insula, visual and somatosensory association areas, cingulate gyrus, prefrontal cortex, as well as subcortical regions, contributing to EF. This study intended to determine associations between EF deterioration during MS and cerebral MS-associated lesion sites. In 31 men with MS (mean age 38.2 ± 11.2 years), we evaluated MS-related EF deterioration by comparing scores of the 5-item International Index of Erectile Function-5 questionnaire (IIEF5) at the time of study and retrospectively, 3 months prior to MS diagnosis, by calculating score differences as DeltaIIEF5 (DeltaIIEF5 score < 0 indicated EF deterioration). To assess the impact of confounding factors of EF, patient age, disease duration, disease severity, depressiveness, bladder and bowel symptoms, and total cerebral MS lesion volume were correlated with DeltaIIEF5 scores (Spearman rank correlation) and compared between patients with and without EF deterioration (t tests or Mann-Whitney U test). MS lesions were assessed on T2-weighted magnetic resonance imaging (MRI; 1.5 or 3 T). We determined the lesion overlap (prevalence of identical lesion sites among patients), subtracted lesion overlaps in patients without EF deterioration from overlaps in patients with EF deterioration, and compared DeltaIIEF5 scores voxel-wise between patients with and without lesions in a given voxel (t test; significance: p < 0.05). In 14 patients (45.2%), DeltaIIEF5 scores indicated EF deterioration. DeltaIIEF5 scores were not associated with age (ρ = 0.06; p = 0.74), disease duration (ρ = 0.26; p = 0.15), disease severity (ρ = - 0.19; p = 0.31), depressiveness (ρ = 0.07; p = 0.72), bladder symptoms (ρ = - 0.11; p = 0.57), bowel symptoms (ρ = 0.17; p = 0.37), and total lesion volume (ρ = - 0.13; p = 0.47). The voxel-wise analysis showed associations between EF deterioration and MS lesions primarily in the bilateral, and predominantly left juxtacortical insular region. In conclusion, MS lesions particularly in the left insular region, which is activated with sexual arousal, contribute to erectile dysfunction.

Background: In women with multiple sclerosis (MS), depression and sexual dysfunction (SD) are common. Whether SD promotes depression or vice versa remains unclear despite therapeutic relevance. Therefore, we aimed to assess whether SD more likely triggers depression or vice versa., Methods: In 83 female MS patients and 21 age-matched healthy women, we assessed depression, using the Beck Depression Inventory-V (BDI-V), and SD using the Female Sexual Function Index (FSFI). We diagnosed depression with BDI-V-scores >35 and SD with FSFI scores < 26.55. We divided patients into groups with and without SD, with and without depression. Between groups, we compared prevalence of SD and depression (Fisher's-exact-test), age, MS-duration, MS-severity, BDI-V-, and FSFI scores (Mann-Whitney U-test; significance: p < 0.05)., Results: A total of 37/83 MS patients and 1/21 controls had SD; 28/83 patients and 3/21 controls had depression; 51.4% patients with SD but only 19.6% without SD had depression (p = 0.003). SD was present in 67.9% depressed and 32.7% non-depressed patients. BDI-V-scores were higher in patients with SD than in patients without SD. FSFI scores were lower in depressed than non-depressed patients., Conclusion: In conclusion, SD was more common than depression. SD afflicted 67.9% depressed MS patients and was also more common in non-depressed MS patients than controls. SD may occur independently from depression while increased depressiveness seems linked to coexistent SD., (© 2018 S. Karger AG, Basel.)

After traumatic brain injury (TBI), central autonomic dysfunction might contribute to long-term increased mortality rates. Central autonomic dysfunction might depend on initial trauma severity. This study was performed to evaluate differences in autonomic modulation at rest and upon standing between patients with a history of mild TBI (post-mild-TBI patients), moderate or severe TBI (post-moderate-severe-TBI patients), and healthy controls. In 20 post-mild-TBI patients (6-78 months after TBI), age-matched 20 post-moderate-severe-TBI patients (6-94 months after TBI) and 20 controls, we monitored respiration, RR intervals (RRI) and systolic blood pressure (BPsys) at supine rest and upon standing. We determined mainly sympathetic low (LF) and parasympathetic high (HF) frequency powers of RRI fluctuations, sympathetically mediated LF-BPsys powers, LF/HF-RRI ratios, normalized (nu) LF-RRI and HF-RRI powers, and compared data between groups, at rest and upon standing (ANOVA with post hoc testing). We correlated autonomic parameters with initial Glasgow Coma Scale (GCS) scores (Spearman test; significance: p < 0.05). Supine BPsys and LFnu-RRI powers were higher while HFnu-RRI powers were lower in post-moderate-severe-TBI patients than post-mild-TBI patients and controls. LFnu-RRI powers were higher and HFnu-RRI powers were lower in post-mild-TBI patients than controls. Upon standing, only post-mild-TBI patients and controls increased LF-BPsys powers and BPsys and decreased HF-RRI powers. GCS scores correlated positively with LFnu-RRI powers, LF/HF-RRI ratios, and inversely with HFnu-RRI powers, at standing position. More than 6 months after TBI, there is autonomic dysfunction at rest and upon standing which is more pronounced after moderate-severe than mild TBI and in part correlates with initial trauma severity.

Acute ischaemic stroke in brain areas contributing to male sexual function may impair erectile function depending on the lesion site. This study intended to determine associations between stroke-related erectile dysfunction and cerebral ischaemic lesion sites using voxel-based lesion mapping. In 52 males (mean age 60.5 ± 10.5 years) with first-ever ischaemic strokes, we assessed erectile function after and retrospectively 3 months prior to the stroke using scores of the 5-item International Index of Erectile Function-5 questionnaire. We assessed cardiovascular risk factors and determined clinical stroke severity and infarct volumes as well as total brain volume by neuroimaging. We calculated correlations between patient age, clinical stroke severity, infarct volumes as well as brain volumes and the difference between erectile dysfunction scores before and after stroke. Moreover, we compared patient age, prevalence of cardiovascular risk factors, clinical stroke severity, infarct volumes and brain volumes of patients with unchanged and deteriorated erectile function after stroke. The infarcts were manually outlined and transformed into stereotaxic space. We determined the lesion overlap and performed subtraction analyses of lesions. In a voxel-based lesion analysis, the difference between erectile dysfunction scores before and after stroke was correlated with the lesion site using t-test statistics. Finally, we conducted a region of interest-based multivariate linear regression analysis that was adjusted for potential confounding factors including patient age, clinical stroke severity, imaging modality, lesion size and brain volume. In 32 patients (61.5%) erectile dysfunction scores declined after the stroke and therefore had stroke-related erectile dysfunction. Deterioration of erectile dysfunction scores was not associated with patient age, clinical stroke severity, infarct volume, brain volume, and cardiovascular risk factors. The voxel-wise subtraction analysis showed associations between stroke-related erectile dysfunction and lesion sites in the right occipito-parietal cortex and thalamus, as well as in the left insula and adjacent temporo-parietal areas. Using voxel-wise t-test statistics, we showed associations between deterioration of erectile function and lesion sites in the right occipital and thalamic region, and the left parietal association area. The linear regression analysis showed that stroke-related erectile dysfunction remained associated with lesions of the right occipital and left parietal association areas after adjusting for confounding factors. In conclusion, our voxel-wise analysis indicates that deteriorating erectile function after stroke is associated with lesions in the right occipito-parietal and thalamic areas integrating visual and somatosensory information, as well as lesions in the left insular and adjacent parieto-temporal areas contributing to generating and mapping visceral arousal states., (© The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Fingolimod slows heart rate (HR) due to vagomimetic effects and might cause additional cardiovascular autonomic changes. While the time course of HR changes is well described, the extent and course of cardiovascular autonomic changes upon fingolimod initiation has not yet been evaluated. This study, therefore, intended to assess cardiovascular autonomic changes during the first 6 h after fingolimod initiation., Methods: In 21 patients with relapsing-remitting multiple sclerosis (RRMS), we recorded respiration (RESP), electrocardiographic RR interval (RRI), systolic and diastolic blood pressure (BPsys, BPdia) at rest, before and 0.5, 1, 2, 3, 4, 5, and 6 h after fingolimod initiation. We calculated parameters of total autonomic modulation [RRI standard deviation (RRI-SD), RRI coefficient of variation (RRI-CV), RRI-total powers], mainly sympathetic cardiac modulation [RRI low frequency (LF) powers], sympathetic BP modulation (BPsys-LF powers), parasympathetic modulation [square root of the mean squared difference of successive RRIs (RMSSD), RRI high frequency (HF) powers], sympatho-vagal cardiac balance (RRI-LF/HF ratios), and baroreflex sensitivity (BRS). We compared parameters between the eight measurements [analysis of variance (ANOVA) or Friedman test with post-hoc analysis; significance: p < 0.05]., Results: After fingolimod initiation, RESP, BPsys, and BPsys-LF powers remained unchanged while RRIs, RRI-CV, RRI-SD, RRI-total powers, RRI-LF powers, RMSSD, RRI-HF powers, and BRS increased after 1 h and rose to peak values occurring after 5, 1, 2, 2, 1, 4, 4, and 4 h, respectively. After 3 h, BPdia had decreased significantly and was lowest after 5 h. RRI-LF/HF ratios decreased to a nadir after 4 h., Conclusions: The increases in parasympathetic and overall cardiac autonomic modulation and in BRS seen with fingolimod initiation are theoretically beneficial for the MS patient's cardiovascular system. However, long-term studies must show whether these effects persist or are attenuated (e.g. due to S1P1 receptor down-regulation upon continued fingolimod therapy)., Competing Interests: Conflict of interest statement: The authors declared the following potential conflicts of interest with respect to the research, authorship, or publication of this article: MJH has received personal compensation for activities from Genzyme, A Sanofi Company (Germany). MJH has received research support from the Rolf and Hubertine Schiffbauer Foundation, Bayer Health Care (Germany) and Novartis Pharma GmbH, Germany. CRL has received travel grants from Genzyme, A Sanofi Company. FC has received travel grants from Genzyme, A Sanofi Company. DHL has received travel grants or speaker honoraria from Bayer Health Care Pharmaceuticals (NJ, USA), Biogen Idec (Cambridge, MA, USA), Merck Serono (Germany), Novartis Pharma GmbH, and TEVA Pharmaceutical Industries (Germany). RAL has received travel grants or speaker honoraria from Bayer Health Care Pharmaceuticals, Biogen Idec, Merck Serono, Novartis Pharma GmbH, Roche (Switzerland), and TEVA Pharmaceutical Industries Ltd. RAL has received research support from Novartis Pharma GmbH, Biogen Idec, and Merck Serono. RW, ML, SR, KHM and KW declare that there is no conflict of interest.

Objective: This study intended to determine associations between alterations of female sexual arousal as well as vaginal lubrication and the site of cerebral multiple sclerosis (MS) lesions., Methods: In 44 women with MS (mean age: 36.5 ± 9.9 years), we assessed their medical history and evaluated sexual function using the Female Sexual Function Index scores for arousal and vaginal lubrication. We determined potential confounding factors of sexual dysfunction: age; disease duration; physical disability; depression; bladder or urinary dysfunction; and total volume of cerebral lesions. Arousal and lubrication scores were correlated with one another and with potential confounding factors. Cerebral MS lesions were recorded on imaging scans. A voxel-based lesion symptom mapping (VLSM) analysis adjusted for confounding variables was performed correlating cerebral sites of MS lesions with arousal and lubrication scores., Results: Decreased arousal scores correlated with decreased lubrication scores; decreased lubrication scores were associated with bladder or urinary symptoms. Arousal and lubrication scores were not associated with any other variables. Multivariate VLSM analysis, including arousal and lubrication scores as covariables of interest, showed right occipital lesions associated with impaired arousal and left insular lesions associated with decreased lubrication. Impaired lubrication remained associated with left insular lesions after adjustment for bladder or urinary dysfunction., Interpretation: Our data indicate that impaired female sexual arousal is associated with MS lesions in the occipital region, integrating visual information and modulating attention toward visual input. Impaired lubrication correlated with lesions in the left insular region, contributing to mapping and generating visceral arousal states. Ann Neurol 2016;80:490-498., (© 2016 American Neurological Association.)

Neuroimaging identified brain areas involved in female orgasm. In women with multiple sclerosis (MS), associations between orgasmic function and the site and size of MS-related magnetic resonance imaging (MRI) changes are undetermined. This study intended to correlate MS-associated cerebral lesion load and location with clinical scores of female orgasmic function. In 50 women with MS (mean age 37.0 ± 9.9 years), we assessed Female Sexual Function Index (FSFI) scores for orgasmic frequency, difficulty and satisfaction. We determined disease duration, Expanded Disability Status Scale (EDSS) scores, and cerebral MS-lesion load and location using T2-weighed 1.5 T MRIs. We correlated FSFI scores for orgasm with patient age, disease duration, EDSS scores, and cerebral MS-lesion load (Spearman rank correlation; significance: p < 0.05). FSFI scores for orgasm correlated inversely with MS-lesion load in the left temporal periventricular white matter and right middle-inferior occipital area, but directly with MS-lesion load in the right frontal primary motor cortex, left prefrontal/inferior frontal cortex, right amygdala, left temporal middle-inferior and fusiform areas, and midbrain. FSFI scores for orgasm did not correlate with patient age, disease duration and EDSS scores (p > 0.05). In conclusion, our results indicate that MS-lesions in left temporal periventricular and right visual association areas deteriorate orgasmic function. In contrast, direct correlations between frontotemporal or midbrain lesions and higher FSFI scores, indicating enhanced or disinhibited orgasmic function, suggest that these brain regions normally buffer orgasmic responses. Moreover, our results indicate that orgasmic dysfunction in women with MS evolves independently of disease duration and physical disability.

Neurocardiological interactions can cause severe cardiac arrhythmias in patients with acute ischemic stroke. The relationship between the lesion location in the brain and the occurrence of cardiac arrhythmias is still discussed controversially. The aim of the present study was to correlate the lesion location with the occurrence of cardiac arrhythmias in patients with acute ischemic stroke. Cardiac arrhythmias were systematically assessed in patients with acute ischemic stroke during the first 72 h after admission to a monitored stroke unit. Voxel-based lesion-symptom mapping (VLSM) was used to correlate the lesion location with the occurrence of clinically relevant severe arrhythmias. Overall 150 patients, 56 with right-hemispheric and 94 patients with a left-hemispheric lesion, were eligible to be included in the VLSM study. Severe cardiac arrhythmias were present in 49 of these 150 patients (32.7%). We found a significant association (FDR correction, q < 0.05) between lesions in the right insular, right frontal and right parietal cortex as well as the right amygdala, basal ganglia and thalamus and the occurrence of cardiac arrhythmias. Because left- and right-hemispheric lesions were analyzed separately, the significant findings rely on the 56 patients with right-hemispheric lesions. The data indicate that these areas are involved in central autonomic processing and that right-hemispheric lesions located to these areas are associated with an elevated risk for severe cardiac arrhythmias.

Objective: A study was undertaken to determine associations between ischemic stroke sites and poststroke hyperglycemia (PSH)., Methods: Nondiabetic patients with first ever ischemic stroke confirmed by imaging were prospectively included. Blood glucose level (BGL), National Institute of Health Stroke Scale (NIHSS) score, and clinical parameters were assessed on admission. BGL was dichotomized for elevated versus normal levels using a cutoff value of >7.0 mmol/l. Clinical parameters were correlated with BGL and were compared between patient groups with elevated versus normal glucose values. A voxel-based lesion symptom mapping (VLSM) analysis adjusted for confounding variables was performed correlating sites of ischemic lesions with PSH., Results: Of 1,281 stroke patients screened, 229 (mean age = 66.3 ± 15.9 years) met the inclusion criteria. Patients with elevated BGL were older, had higher NIHSS scores, and had larger infarcts compared to those without elevated glucose levels. Spearman rank analysis showed correlations between BGL and age, infarct size, heart rate (HR), and NIHSS scores (p ≤ 0.05). The VLSM analysis adjusted for these confounding factors demonstrated associations between PSH and damaged voxels in right hemispheric insular and opercular areas., Interpretation: The data indicate that damage in the right insulo-opercular areas contributes to PSH. The association between sympathetically mediated increase of HR and BGL suggests disinhibition of sympathetic outflow as a possible mechanism for PSH., (© 2014 American Neurological Association.)

Background: Structural analysis of biochemical networks is a growing field in bioinformatics and systems biology. The availability of an increasing amount of biological data from molecular biological networks promises a deeper understanding but confronts researchers with the problem of combinatorial explosion. The amount of qualitative network data is growing much faster than the amount of quantitative data, such as enzyme kinetics. In many cases it is even impossible to measure quantitative data because of limitations of experimental methods, or for ethical reasons. Thus, a huge amount of qualitative data, such as interaction data, is available, but it was not sufficiently used for modeling purposes, until now. New approaches have been developed, but the complexity of data often limits the application of many of the methods. Biochemical Petri nets make it possible to explore static and dynamic qualitative system properties. One Petri net approach is model validation based on the computation of the system's invariant properties, focusing on t-invariants. T-invariants correspond to subnetworks, which describe the basic system behavior.With increasing system complexity, the basic behavior can only be expressed by a huge number of t-invariants. According to our validation criteria for biochemical Petri nets, the necessary verification of the biological meaning, by interpreting each subnetwork (t-invariant) manually, is not possible anymore. Thus, an automated, biologically meaningful classification would be helpful in analyzing t-invariants, and supporting the understanding of the basic behavior of the considered biological system., Methods: Here, we introduce a new approach to automatically classify t-invariants to cope with network complexity. We apply clustering techniques such as UPGMA, Complete Linkage, Single Linkage, and Neighbor Joining in combination with different distance measures to get biologically meaningful clusters (t-clusters), which can be interpreted as modules. To find the optimal number of t-clusters to consider for interpretation, the cluster validity measure, Silhouette Width, is applied., Results: We considered two different case studies as examples: a small signal transduction pathway (pheromone response pathway in Saccharomyces cerevisiae) and a medium-sized gene regulatory network (gene regulation of Duchenne muscular dystrophy). We automatically classified the t-invariants into functionally distinct t-clusters, which could be interpreted biologically as functional modules in the network. We found differences in the suitability of the various distance measures as well as the clustering methods. In terms of a biologically meaningful classification of t-invariants, the best results are obtained using the Tanimoto distance measure. Considering clustering methods, the obtained results suggest that UPGMA and Complete Linkage are suitable for clustering t-invariants with respect to the biological interpretability., Conclusion: We propose a new approach for the biological classification of Petri net t-invariants based on cluster analysis. Due to the biologically meaningful data reduction and structuring of network processes, large sets of t-invariants can be evaluated, allowing for model validation of qualitative biochemical Petri nets. This approach can also be applied to elementary mode analysis.