Pharmacological preclinical comparison of tenecteplase and alteplase for the treatment of acute stroke.

Alteplase (rtPA) remains the standard thrombolytic drug for acute ischemic stroke. However, new rtPA-derived molecules, such as tenecteplase (TNK), with prolonged half-lives following a single bolus administration, have been developed. Although TNK is currently under clinical evaluation, the limited preclinical data highlight the need for additional studies to elucidate its benefits. The toxicities of rtPA and TNK were evaluated in endothelial cells, astrocytes, and neuronal cells. In addition, their in vivo efficacy was independently assessed at two research centers using an ischemic thromboembolic mouse model. Both therapies were tested via early (20 and 30 min) and late administration (4 and 4.5 h) after stroke. rtPA, but not TNK, caused cell death only in neuronal cultures. Mice were less sensitive to thrombolytic therapies than humans, requiring doses 10-fold higher than the established clinical dose. A single bolus dose of 2.5 mg/kg TNK led to an infarct reduction similar to perfusion with 10 mg/kg of rtPA. Early administration of TNK decreased the hemorrhagic transformations compared to that by the early administration of rtPA; however, this result was not obtained following late administration. These two independent preclinical studies support the use of TNK as a promising reperfusion alternative to rtPA.

New Perspectives in Neuroprotection for Ischemic Stroke.

The constant failure of new neuroprotective therapies for ischemic stroke has partially halted the search for new therapies in recent years, mainly because of the high investment risk required to develop a new treatment for a complex pathology, such as stroke, with a narrow intervention window and associated comorbidities. However, owing to recent progress in understanding the stroke pathophysiology, improvement in patient care in stroke units, development of new imaging techniques, search for new biomarkers for early diagnosis, and increasingly widespread use of mechanical recanalization therapies, new opportunities have opened for the study of neuroprotection. This review summarizes the main protective agents currently in use, some of which are already in the clinical evaluation phase. It also includes an analysis of how recanalization therapies, new imaging techniques, and biomarkers have improved their efficacy.

Neuroprotection Afforded by an Enriched Mediterranean-like Diet Is Modified by Exercise in a Rat Male Model of Cerebral Ischemia.

Ischemic stroke is an important cause of mortality and disability worldwide. Given that current treatments do not allow a remarkably better outcome in patients after stroke, it is mandatory to seek new approaches to preventing stroke and/or complementing the current treatments or ameliorating the ischemic insult. Multiple preclinical and clinical studies highlighted the potential beneficial roles of exercise and a Mediterranean diet following a stroke. Here, we investigated the effects of a pre-stroke Mediterranean-like diet supplemented with hydroxytyrosol and with/without physical exercise on male rats undergoing transient middle cerebral artery occlusion (tMCAO). We also assessed a potential synergistic effect with physical exercise. Our findings indicated that the diet reduced infarct and edema volumes, modulated acute immune response by altering cytokine and chemokine levels, decreased oxidative stress, and improved acute functional recovery post-ischemic injury. Interestingly, while physical exercise alone improved certain outcomes compared to control animals, it did not enhance, and in some aspects even impaired, the positive effects of the Mediterranean-like diet in the short term. Overall, these data provide the first preclinical evidence that a preemptive enriched Mediterranean diet modulates cytokines/chemokines levels downwards which eventually has an important role during the acute phase following ischemic damage, likely mediating neuroprotection.

Perfusion-weighted software written in Python for DSC-MRI analysis.

Introduction: Dynamic susceptibility-weighted contrast-enhanced (DSC) perfusion studies in magnetic resonance imaging (MRI) provide valuable data for studying vascular cerebral pathophysiology in different rodent models of brain diseases (stroke, tumor grading, and neurodegenerative models). The extraction of these hemodynamic parameters via DSC-MRI is based on tracer kinetic modeling, which can be solved using deconvolution-based methods, among others. Most of the post-processing software used in preclinical studies is home-built and custom-designed. Its use being, in most cases, limited to the institution responsible for the development. In this study, we designed a tool that performs the hemodynamic quantification process quickly and in a reliable way for research purposes. Methods: The DSC-MRI quantification tool, developed as a Python project, performs the basic mathematical steps to generate the parametric maps: cerebral blood flow (CBF), cerebral blood volume (CBV), mean transit time (MTT), signal recovery (SR), and percentage signal recovery (PSR). For the validation process, a data set composed of MRI rat brain scans was evaluated: i) healthy animals, ii) temporal blood-brain barrier (BBB) dysfunction, iii) cerebral chronic hypoperfusion (CCH), iv) ischemic stroke, and v) glioblastoma multiforme (GBM) models. The resulting perfusion parameters were then compared with data retrieved from the literature. Results: A total of 30 animals were evaluated with our DSC-MRI quantification tool. In all the models, the hemodynamic parameters reported from the literature are reproduced and they are in the same range as our results. The Bland-Altman plot used to describe the agreement between our perfusion quantitative analyses and literature data regarding healthy rats, stroke, and GBM models, determined that the agreement for CBV and MTT is higher than for CBF. Conclusion: An open-source, Python-based DSC post-processing software package that performs key quantitative perfusion parameters has been developed. Regarding the different animal models used, the results obtained are consistent and in good agreement with the physiological patterns and values reported in the literature. Our development has been built in a modular framework to allow code customization or the addition of alternative algorithms not yet implemented.

Mild systemic inflammation enhances response to OnabotulinumtoxinA in chronic migraineurs.

The anti-inflammatory effect of OnabotulinumtoxinA (OnabotA) has been a matter of discussion for many years. In chronic migraine, however, increased pro-inflammatory state is associated with good response to OnabotA. We aimed to investigate whether a mild systemic inflammatory state elicited by a common oral infection (periodontitis) could enhance treatment response to OnabotA. In this study, we included 61 chronic migraineurs otherwise healthy treated with OnabotA of which 7 were poor responders and 54 good responders. Before receiving OnabotA therapy, all participants underwent a full-mouth periodontal examination and blood samples were collected to determine serum levels of calcitonin gene-related peptide (CGRP), interleukin 6 (IL-6), IL-10 and high sensitivity C-reactive protein (hs-CRP). Periodontitis was present in 70.4% of responders and 28.6% of non-responders (P = 0.042). Responders showed greater levels of inflammation than non-responders (IL-6: 15.3 ± 8.7 vs. 9.2 ± 4.7 ng/mL, P = 0.016; CGRP: 18.8 ± 7.6 vs. 13.0 ± 3.1 pg/mL, P = 0.002; and hs-CRP: 3.9 ± 6.6 vs. 0.9 ± 0.8 mg/L, P = 0.003). A linear positive correlation was found between the amount of periodontal tissue inflamed in the oral cavity and markers of inflammation (IL-6: r = 0.270, P = 0.035; CGRP: r = 0.325, P = 0.011; and hs-CRP: r = 0.370, P = 0.003). This report shows that in presence of elevated systemic inflammatory markers related to periodontitis, OnabotA seems to reduce migraine attacks. The changes of scheduled inflammatory parameters after treatment and subsequent assessment during an adequate period still need to be done.

[18F]-FMISO PET/MRI Imaging Shows Ischemic Tissue around Hematoma in Intracerebral Hemorrhage.

Intracerebral hemorrhage (ICH), being the most severe cerebrovascular disease, accounts for 10-15% of all strokes. Hematoma expansion is one of the most important factors associated with poor outcome in intracerebral hemorrhage (ICH). Several studies have suggested that an "ischemic penumbra" might arise when the hematoma has a large expansion, but clinical studies are inconclusive. We performed a preclinical study to demonstrate the presence of hypoxic-ischemic tissue around the hematoma by means of longitudinal [18F]-fluoromisonidazole ([18F]-FMISO) PET/MRI studies over time in an experimental ICH model. Our results showed that all [18F]-FMISO PET/MRI images exhibited hypoxic-ischemic tissue around the hematoma area. A significant increase of [18F]-FMISO uptake was found at 18-24 h post-ICH when the maximum of hematoma volume is achieved and this increase disappeared before 42 h. These results demonstrate the presence of hypoxic tissue around the hematoma and open the possibility of new therapies aimed to reduce ischemic damage associated with ICH.

Severe periodontitis is linked with increased peripheral levels of sTWEAK and PTX3 in chronic migraineurs.

OBJECTIVES: Periodontitis (PD) and chronic migraine (CM) have been recently linked, and inflammatory processes and vascular endothelial changes are hypothesized as potential mediators of this relationship. The aim of this cross-sectional analysis was to investigate the potential association of PD with vascular systemic inflammation and complement activation in patients with CM. MATERIALS AND METHODS: Ninety-four chronic migraineurs underwent a full-mouth periodontal evaluation and a measure of PD activity and severity, namely the periodontal inflamed surface area (PISA) was calculated for each patient. We divided CM patients according to their periodontal status: mild PD (N = 14), moderate PD (N = 22), severe PD (N = 19), and non-PD (N = 39). Serum levels of C-reactive protein (CRP), pentraxin 3 (PTX3), soluble tumor necrosis factor-like weak inducer of apoptosis (sTWEAK), and complements C3 and C4 were measured outside of migraine attacks. RESULTS: We found that severe periodontal patients had significantly higher circulating levels of PTX3 and sTWEAK compared with those without PD (2475.3 ± 1646.8 pg/mL vs. 516.6 ± 1193.8 pg/mL, P < 0.0001 and 672.4 ± 118.2 pg/mL vs. 485.7 ± 112.2 pg/mL, P < 0.0001; respectively). For the remaining biomarkers, no significant differences were found between groups. Severe PD was independently associated with higher levels of PTX3 (ß = 1997.6, P < 0.0001) and sTWEAK (ß = 187.1, P < 0.0001) but not with CRP, C3, and C4. PISA positively correlated to PTX3 (r = 0.475, P < 0.0001) and sTWEAK (r = 0.386, P < 0.0001). CONCLUSIONS: Based on these preliminary results, severe PD was linked with vascular systemic inflammation in patients with CM. However, further longitudinal studies should be performed to confirm these findings. CLINICAL RELEVANCE: sTWEAK and PTX3 measured in serum could be used as biomarkers in the PD-CM link.

Light-Emitting Diode Photobiomodulation After Cerebral Ischemia.

Photobiomodulation (PBM) therapy is a promising therapeutic approach for several pathologies, including stroke. The biological effects of PBM for the treatment of cerebral ischemia have previously been explored as a neuroprotective strategy using different light sources, wavelengths, and incident light powers. However, the capability of PBM as a novel alternative therapy to stimulate the recovery of the injured neuronal tissue after ischemic stroke has been poorly explored. The aim of this study was to investigate the low-level light irradiation therapy by using Light Emitting Diodes (LEDs) as potential therapeutic strategy for stroke. The LED photobiomodulation (continuous wave, 830 nm, 0.2-0.6 J/cm2) was firstly evaluated at different energy densities in C17.2 immortalized mouse neural progenitor cell lines, in order to observe if this treatment had any effect on cells, in terms of proliferation and viability. Then, the PBM-LED effect (continuous wave, 830 nm, 0.28 J/cm2 at brain cortex) on long-term recovery (12 weeks) was analyzed in ischemic animal model by means lesion reduction, behavioral deficits, and functional magnetic resonance imaging (fMRI). Analysis of cellular proliferation after PBM was significantly increased (1 mW) in all different exposure times used; however, this effect could not be replicated in vivo experimental conditions, as PBM did not show an infarct reduction or functional recovery. Despite the promising therapeutic effect described for PBM, further preclinical studies are necessary to optimize the therapeutic window of this novel therapy, in terms of the mechanism associated to neurorecovery and to reduce the risk of failure in futures clinical trials.

Periodontal inflammation is related to increased serum calcitonin gene-related peptide levels in patients with chronic migraine.

BACKGROUND: Recently, a relationship was found between periodontitis and chronic migraine. Calcitonin gene-related peptide (CGRP) is a key element in migraine pathophysiology. However, no information exists of the potential association between periodontal inflammation and CGRP in chronic migraine. The aim of the study was, therefore, to investigate whether there is a link between periodontitis and peripheral levels of CGRP in a cohort of patients with chronic migraine. METHODS: We included 102 chronic migraineurs and 77 age- and sex-matched individuals free of headache/migraine. Full-mouth periodontal parameters were recorded and the periodontal inflamed surface area (PISA) was calculated to quantify the periodontal inflammatory status for each participant. Sociodemographic data and comorbidities were assessed by means of a standard questionnaire. We collected blood samples and serum concentrations were done for CGRP, interleukin (IL)-6 and IL-10. RESULTS: In the chronic migraine group, patients with periodontitis had greater levels of serum CGRP (19.7 ± 6.5 versus 15.3 ± 6.2 pg/mL, P < 0.0001) and IL-6 (15.1 ± 9.2 versus 9.6 ± 6.3 pg/mL, P < 0.0001) while non-significant differences were observed with IL-10 (2.0 ± 1.0 versus 2.8 ± 1.5 pg/mL, P = 0.675) concentrations than those without periodontitis. PISA was independently associated with CGRP in patients with chronic migraine (ß = 0.003; 95% confidence interval: 0.001 to 0.006, P = 0.031). PISA correlated positively with CGRP (r = 0.236; P = 0.017) and IL-6 (r = 0.262; P = 0.008) in chronic migraine. CONCLUSIONS: Periodontal inflammation is associated with increased circulating levels of CGRP in chronic migraineurs. Elucidating the exact mechanisms through which periodontitis and CGRP are linked in these patients deserves further investigation.

Trends in stroke outcomes in the last ten years in a European tertiary hospital.

BACKGROUND: Studying the impact of demographic changes and progress in the management of stroke patients is necessary in order to organize care structures for the coming years. Consequently, we analyzed the prognostic trends of patients admitted to the Stroke Unit of a tertiary hospital in the last ten years. METHODS: The University Clinical Hospital of Santiago de Compostela is the referral hospital for stroke in a catchment area that accounts for 16.5% of the population of Galicia. Data from patients admitted to the Stroke Unit were registered prospectively. A multinomial logistic regression was performed to determine the influence of new trends in demographic factors and in the management of patients with acute stroke. For the expected trend of progression, a 2008-2011 and 2012-2017 time series model was made by selecting the most appropriate model. RESULTS: In the last 10 years, the age of stroke onset has only increased in women (from 74.4 ± 2.2 years in 2008 to 78.8 ± 2.1 years in 2017; p = 0.037), and the same happens with the severity of neurological symptoms (ischemic stroke (IS), p < 0.0001; from 14 [10, 19] in 2008 to 19 [15, 26] in 2017), with a higher percentage of cardioembolic strokes (40.7% vs. 32.2% of cardioembolic strokes in women vs. men, p < 0.0001). In a multiple linear regression model, hospital improvement was mainly associated with the use of reperfusion treatment (B 53.11, CI 95% 49.87, 56.36, p < 0.0001). A differentiated multinomial logistic regression analysis conducted for the whole sample with ischemic strokes in the two time periods (2008-2011 and 2012-2017) showed no differences in the influence of factors associated with higher morbidity and mortality. The modeling of time series showed a distinct falling trend in mortality, with a slight increase in good outcome as well as morbidity in both ischemic and hemorrhagic stroke. CONCLUSIONS: Our results showed that mortality decreased in the entire sample; however, although outcome at discharge improved in ischemic stroke, severe disability also increased in these patients. Importantly, this tendency towards increased morbidity seems to be confirmed for the coming years.

Generation and characterization of the human iPSC line IDISi001-A isolated from blood cells of a CADASIL patient carrying a NOTCH3 mutation.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common form of hereditary stroke disorder. It is caused by mutations in NOTCH3 that lead to progressive degeneration of the smooth muscle cells in blood vessels. There is currently no treatment for this disorder. We reprogrammed to pluripotency blood mononuclear cells isolated from a patient carrying a NOTCH3 mutation by using a commercially available non-integrating system. The success in the generation of this iPSC line (IDISi001-A) suggests that the NOTCH3 mutation did not limit cell reprogramming and offers an unprecedented opportunity for studying and modeling CADASIL pathology.

CM352 Reduces Brain Damage and Improves Functional Recovery in a Rat Model of Intracerebral Hemorrhage.

BACKGROUND: Intracerebral hemorrhage (ICH) is an acute neurological disorder with high mortality and no effective treatment. In addition to the initial bleeding event, rebleeding and hematoma expansion are associated with poor outcome in these patients. We studied the effectiveness of the new antifibrinolytic agent CM352, a short-half-life matrix metalloproteinase inhibitor, for achieving early hemostasis and improving functional recovery in a rat model of collagenase-induced ICH. METHODS AND RESULTS: ICH was induced by striatal injection of collagenase, and 1 hour later, rats received an intravenous injection of saline (n=6) or CM352 (1 mg/kg, n=6). Hematoma (basal and after 3 and 24 hours) and lesion (14 days) volumes were quantified on T2-weighted (T2) magnetic resonance images. Neurological and functional recovery was evaluated by using Bederson score and a cylinder test (basal, 24 hours, and 14 days). Early treatment (1 hour) with CM352 was efficient reducing hematoma expansion at 3 hours (P<0.01) and, more markedly, at 24 hours (P<0.01). Decreased bleeding after antifibrinolytic treatment was accompanied by reduced interleukin-6 levels at 3 hours (P<0.05) and smaller lesion volume at 14 days (P<0.01). CM352 drastically reduced sensorimotor impairment (cylinder test) after ICH in rats at 24 hours (P<0.01) and 14 days (P<0.01). Similarly, it also attenuated neurological deficit (Bederson scale) at 24 hours (P<0.01) and 14 days (P<0.01). Interestingly, late (3 hours) CM352 administration also resulted in reduced lesion size and better functional outcome. CONCLUSIONS: CM352, a new antifibrinolytic agent and matrix metalloproteinase inhibitor, effectively prevented hematoma growth and reduced lesion size in ICH in association with improved functional and neurological recovery.

Vectorized nanodelivery systems for ischemic stroke: a concept and a need.

Neurological diseases of diverse aetiologies have significant effects on the quality of life of patients. The limited self-repairing capacity of the brain is considered to be the origin of the irreversible and progressive nature of many neurological diseases. Therefore, neuroprotection is an important goal shared by many clinical neurologists and neuroscientists. In this review, we discuss the main obstacles that have prevented the implementation of experimental neuroprotective strategies in humans and propose alternative avenues for the use of neuroprotection as a feasible therapeutic approach. Special attention is devoted to nanotechnology, which is a new approach for developing highly specific and localized biomedical solutions for the study of the multiple mechanisms involved in stroke. Nanotechnology is contributing to personalized neuroprotection by allowing us to identify mechanisms, determine optimal therapeutic windows, and protect patients from brain damage. In summary, multiple aspects of these new players in biomedicine should be considered in future in vivo and in vitro studies with the aim of improving their applicability to clinical studies.

Endothelial Progenitor Cells as a Therapeutic Approach for Intracerebral Hemorrhage.

BACKGROUND: Intracerebral hemorrhage (ICH) is the most lethal subtype of stroke, a leading cause of death and disability in developed countries. Therapeutic options are notably limited. There is no specific pharmacological treatment, and early surgery has few indications that represent only a small clinically relevant survival advantage. It is therefore mandatory to investigate repairing processes after ICH in order to develop related therapeutic strategies. METHODS: The goal of this review is to discuss the current status of knowledge about the potential therapeutic role of endothelial progenitor cells (EPCs) in ICH, as well as the possible molecular mechanisms and future perspectives. RESULTS: ICH is characterized by a primary vascular rupture, followed by a secondary vascular tearing due to the peripheral pressure exerted by the hematoma. Hypoperfusion may also play a role, although not as markedly as in ischemic stroke. In this context, the repairing of damaged vessels and the development of new ones seem logical therapeutic targets. Circulating EPCs have been suggested to play a major role in re-endothelization, angiogenesis and vasculogenesis. Congruently, EPC levels have been associated with good neurological and functional outcome as well as with reduced residual volume in patients with acute ICH. CONCLUSION: An EPC-based therapy, acting primarily through angiogenic mechanisms, may be a valid therapeutic option in ICH.

Impact of benzodiazepines on brain FDG-PET quantification after single-dose and chronic administration in rats.

INTRODUCTION: Current guidelines for brain PET imaging advice against the injection of diazepam prior to brain FDG-PET examination in order to avoid possible interactions of benzodiazepines with the radiotracer uptake. Nevertheless, many patients undergoing PET studies are likely to be under chronic treatment with benzodiazepines, for example due to the use of different medications such as sleeping pills. Animal studies may provide an extensive and accurate estimation of the effect of benzodiazepines on brain metabolism in a well-defined and controlled framework. AIM: This study aims at evaluating the impact of benzodiazepines on brain FDG uptake after single-dose administration and chronic treatment in rats. METHODS: Twelve Sprague-Dawley healthy rats were randomly divided into two groups, one treated with diazepam and the other used as control group. Both groups underwent PET/CT examinations after single-dose and chronic administration of diazepam (treated) or saline (controls) during twenty-eight days. Different atlas-based quantification methods were used to explore differences on the total uptake and uptake patterns of FDG between both groups. RESULTS: Our analysis revealed a significant reduction of global FDG uptake after acute (-16.2%) and chronic (-23.2%) administration of diazepam. Moreover, a strong trend pointing to differences between acute and chronic administrations (p<0.08) was also observed. Uptake levels returned to normal after interrupting the administration of diazepam. On the other hand, patterns of FDG uptake were not affected by the administration of diazepam. CONCLUSIONS: The administration of diazepam causes a progressive decrease of the FDG global uptake in the rat brain, but it does not change local patterns within the brain. Under these conditions, visual assessment and quantification methods based on regional differences such as asymmetry indexes or SPM statistical analysis would still be valid when administrating this medication.

Increased Endothelial Progenitor Cell Levels are Associated with Good Outcome in Intracerebral Hemorrhage.

Circulating endothelial progenitor cells (EPCs) play a role in the regeneration of damaged brain tissue. However, the relationship between circulating EPC levels and functional recovery in intracerebral hemorrhage (ICH) has not yet been tested. Therefore, our aim was to study the influence of circulating EPCs on the outcome of ICH. Forty-six patients with primary ICH (males, 71.7%; age, 72.7 ± 10.8 years) were prospectively included in the study within 12 hours of symptom onset. The main outcome variable was good functional outcome at 12 months (modified Rankin scale ≤2), considering residual volume at 6 months as a secondary variable. Circulating EPC (CD34(+)/CD133(+)/KDR(+)) levels were measured by flow cytometry from blood samples obtained at admission, 72 hours and day 7. Our results indicate that patients with good outcome show higher EPC numbers at 72 hours and day 7 (all p < 0.001). However, only EPC levels at day 7 were independently associated with good functional outcome at 12 months (OR, 1.15; CI95%, 1.01-1.35) after adjustment by age, baseline stroke severity and ICH volume. Moreover, EPC levels at day 7 were negatively correlated to residual volume (r = -0.525; p = 0.005). In conclusion, these findings suggest that EPCs may play a role in the functional recovery of ICH patients.