Circulating Monocytes Exhibit an Endotoxin Tolerance Status after Acute Ischemic Stroke: Mitochondrial DNA as a Putative Explanation for Poststroke Infections.

Patients with acute ischemic stroke (AIS) suffer from infections associated with mortality. The relevance of the innate immune system, and monocytes in particular, has emerged as an important factor in the evolution of these infections. The study enrolled 14 patients with AIS, without previous treatment, and 10 healthy controls. In the present study, we show that monocytes from patients with AIS exhibit a refractory state or endotoxin tolerance. The patients were unable to orchestrate an inflammatory response against LPS and expressed three factors reported to control the evolution of human monocytes into a refractory state: IL-1R-associated kinase-M, NFkB2/p100, and hypoxia-inducible factor-1α. The levels of circulating mitochondrial DNA (mtDNA) in patients with AIS correlated with impaired inflammatory response of isolated monocytes. Interestingly, the patients could be classified into two groups: those who were infected and those who were not, according to circulating mtDNA levels. This finding was validated in an independent cohort of 23 patients with AIS. Additionally, monocytes from healthy controls, cultured in the presence of both sera from patients and mtDNA, reproduced a refractory state after endotoxin challenge. This effect was negated by either a TLR9 antagonist or DNase treatment. The present data further extend our understanding of endotoxin tolerance implications in AIS. A putative role of mtDNA as a new biomarker of stroke-associated infections, and thus a clinical target for preventing poststroke infection, has also been identified.

Brain-derived neurotrophic factor administration mediated oligodendrocyte differentiation and myelin formation in subcortical ischemic stroke.

BACKGROUND AND PURPOSE: Translational research is beginning to reveal the importance of trophic factors as a therapy for cellular brain repair. The purpose of this study was to analyze whether brain-derived neurotrophic factor (BDNF) administration could mediate oligodendrogenesis and remyelination after white matter injury in subcortical stroke. METHODS: Ischemia was induced in rats by injection of endothelin-1. At 24 hours, 0.4 µg/kg of BDNF or saline was intravenously administered to the treatment and control groups, respectively. Functional evaluation, MRI, and fiber tract integrity on tractography images were analyzed. Proliferation (KI-67) and white matter repair markers (A2B5, 2',3'-cyclic-nucleotide 3'-phosphodiesterase [CNPase], adenomatous polyposis coli [APC], platelet-derived growth factor receptor alpha [PDGFR-α], oligodendrocyte marker O4 [O4], oligodendrocyte transcription factor [Olig-2], and myelin basic protein [MBP]) were analyzed at 7 and 28 days. RESULTS: The BDNF-treated animals showed less functional deficit at 28 days after treatment than the controls (P<0.05). Although T2-MRI did not show differences in lesion size at 7 and 28 days between groups, diffusion tensor imaging tractography analysis revealed significantly better tract connectivity at 28 days in the BDNF group than in the controls (P<0.05). Increased proliferation of oligodendrocyte progenitors was observed in treated animals at 7 days (P<0.05). Finally, the levels of white matter repair markers (A2B5, CNPase, and O4 at 7 days; Olig-2 and MBP at 28 days) were higher in the BDNF group than in the controls (P<0.05). CONCLUSIONS: BDNF administration exerted better functional outcome, oligodendrogenesis, remyelination, and fiber connectivity than controls in rats subjected to subcortical damage in ischemic stroke.

Different protective and reparative effects of olmesartan in stroke according to time of administration and withdrawal.

Angiotensin type 1 receptor blockers (ARBs) have induced improved functional recovery and reduced infarct volume in experimental animal models of stroke. Clinical data have indicated a positive correlation between prestroke treatment with ARBs and reduced stroke severity and better outcomes; however, the mechanisms of these beneficial effects are not yet well understood. This study compares the protective and possible reparative effects of continuous oral treatment with olmesartan (OLM) with OLM pretreatment and withdrawal after permanent middle cerebral artery occlusion (pMCAO) in rats. Fifty-two Sprague-Dawley rats were randomly assigned to five groups: MCAO(-/OLM) (OLM 10 mg/kg/day for 14 days after infarct), MCAO(OLM/OLM) (OLM 10 mg/kg/day for 7 days before and 14 days after infarct), MCAO(OLM/-) (OLM 10 mg/kg/day for 7 days before infarct), sham, and control. We analyzed functional recovery; lesion size; cell death; expression of the pro-oxidant enzyme NADPH oxidase 4 (NOX-4); isolectin-B4; and repair markers such as glial fibrillary acidic protein, vascular endothelial growth factor (VEGF), and brain-derived neurotrophic factor (BDNF). All of the OLM-treated groups showed significantly better functional scores and reduced infarct sizes and cell death compared with the control group 14 days after pMCAO. Levels of NOX-4, VEGF, and BDNF were significantly lower in the brains of the MCAO(OLM/OLM) and sham groups compared with the other groups. OLM treatment improved functional recovery and reduced lesion size and cell death after cerebral ischemia. Only the continuous administration of OLM before and after stroke reduced oxidative stress levels, with better tissue preservation, without triggering brain repair marker activation.

Comparison between xenogeneic and allogeneic adipose mesenchymal stem cells in the treatment of acute cerebral infarct: proof of concept in rats.

BACKGROUND: Rat adipose tissue-derived-mesenchymal stem cells (rAD-MSCs) have proven to be safe in experimental animal models of stroke. However, in order to use human AD-MSCs (hAD-MSCs) as a treatment for stroke patients, a proof of concept is needed. We analyzed whether the xenogeneic hAD-MSCs were as safe and effective as allogeneic rAD-MSCs in permanent Middle Cerebral Artery Occlusion (pMCAO) in rats. METHODS: Sprague-Dawley rats were randomly divided into three groups, which were intravenously injected with xenogeneic hAD-MSCs (2 × 10(6)), allogeneic rAD-MSCs (2 × 10(6)) or saline (control) at 30 min after pMCAO. Behavior, cell implantation, lesion size and cell death were evaluated. Brain markers such as GFAP (glial fibrillary acid protein), VEGF (vascular endothelial growth factor) and SYP (synaptophysin) and tumor formation were analyzed. RESULTS: Compared to controls, recovery was significantly better at 24 h and continued to be so at 14 d after IV administration of either hAD-MSCs or rAD-MSCs. No reduction in lesion size or migration/implantation of cells in the damaged brain were observed in the treatment groups. Nevertheless, cell death was significantly reduced with respect to the control group in both treatment groups. VEGF and SYP levels were significantly higher, while those of GFAP were lower in the treated groups. At three months, there was no tumor formation. CONCLUSIONS: hAD-MSCs and rAD-MSCs were safe and without side effects or tumor formation. Both treatment groups showed equal efficacy in terms of functional recovery and decreased ischemic brain damage (cell death and glial scarring) and resulted in higher angiogenesis and synaptogenesis marker levels.

Intralesional Patterns of MRI ADC Maps Predict Outcome in Experimental Stroke.

BACKGROUND: After acute ischemia, the tissue that is at risk of infarction can be detected by perfusion-weighted imaging/diffusion-weighted imaging (PWI/DWI) mismatch but the time that is needed to process PWI limits its use. As DWI is highly sensitive to acute ischemic tissue damage, we hypothesized that different ADC patterns represent areas with a different potential for recovery. METHODS: In a model of permanent middle cerebral artery occlusion (pMCAO), Sprague-Dawley rats were randomly distributed to sham surgery and pMCAO. We further separated the pMCAO group according to intralesional ADC pattern (homogeneous or heterogeneous). At 24 h after ischemia induction, we analyzed lesion size, functional outcome, cell death expression, and brain protection markers including ROS enzyme NOX-4. MRI included DWI (ADC maps), DTI (tractography), and PWI (CBF, CBV and MTT). RESULTS: The lesion size was similar in pMCAO rats. Animals with a heterogeneous pattern in ADC maps showed better functional outcome in Rotarod test (p = 0.032), less expression of cell death (p = 0.014) and NOX-4 (p = 0.0063), higher intralesional CBF (p = 0.0026) and larger PWI/DWI mismatch (p = 0.007). CONCLUSIONS: In a rodent model for ischemic stroke, intralesional heterogeneity in ADC maps was related to better functional outcome in lesions of similar size and interval after pMCAO. DWI ADC maps may assist in the early identification of ischemic tissue with an increased potential for recovery as higher expression of acute protection markers, lower expression of cell death, increased PWI/DWI mismatch, and higher intralesional CBF were present in animals with a heterogeneous ADC pattern.

Reparative therapy for acute ischemic stroke with allogeneic mesenchymal stem cells from adipose tissue: a safety assessment: a phase II randomized, double-blind, placebo-controlled, single-center, pilot clinical trial.

BACKGROUND: Few studies have evaluated the possible beneficial effect of the administration of stem cells in the early stages of stroke. Intravenous administration of allogeneic mesenchymal stem cells (MSCs) from adipose tissue in patients with acute stroke could be a safe therapy for promoting neurovascular unit repair, consequently supporting better functional recovery. We aim to assess the safety and efficacy of MSC administration and evaluate its potential as a treatment for cerebral protection and repair. MATERIALS: A Phase IIa, prospective, randomized, double-blind, placebo-controlled, single-center, pilot clinical trial. Twenty patients presenting acute ischemic stroke will be randomized in a 1:1 proportion to treatment with allogeneic MSCs from adipose tissue or to placebo (or vehicle) administered as a single intravenous dose within the first 2 weeks after the onset of stroke symptoms. The patients will be followed up for 2 years. Primary outcomes for safety analysis: adverse events (AEs) and serious AEs; neurologic and systemic complications, and tumor development. Secondary outcomes for efficacy analysis: modified Rankin Scale; NIHSS; infarct size; and biochemical markers of brain repair (vascular endothelial growth factor, brain-derived neurotrophic factor, and matrix metalloproteinases 9). RESULTS AND CONCLUSIONS: To our knowledge, this is the first, phase II, pilot clinical trial to investigate the safety and efficacy of intravenous administration of allogeneic MSCs from adipose tissue within the first 2 weeks of stroke. In addition, its results will help us define the best criteria for a future phase III study.

Trophic factors and cell therapy to stimulate brain repair after ischaemic stroke.

Brain repair involves a compendium of natural mechanisms that are activated following stroke. From a therapeutic viewpoint, reparative therapies that encourage cerebral plasticity are needed. In the last years, it has been demonstrated that modulatory treatments for brain repair such as trophic factor- and stem cell-based therapies can promote neurogenesis, gliogenesis, oligodendrogenesis, synaptogenesis and angiogenesis, all of which having a beneficial impact on infarct volume, cell death and, finally, and most importantly, on the functional recovery. However, even when promising results have been obtained in a wide range of experimental animal models and conditions these preliminary results have not yet demonstrated their clinical efficacy. Here, we focus on brain repair modulatory treatments for ischaemic stroke, that use trophic factors, drugs with trophic effects and stem cell therapy. Important and still unanswered questions for translational research ranging from experimental animal models to recent and ongoing clinical trials are reviewed here.

Exosomes promote restoration after an experimental animal model of intracerebral hemorrhage.

Exosomes are gaining importance because they show great promise in therapeutic applications for several diseases. Particularly in stroke, exosomes derived from mesenchymal stem cell (MSC) therapy work as paracrine effectors responsible for promoting neurovascular remodeling and functional recovery. Adult male rats were subjected to intracerebral hemorrhage (ICH) by intrastriatal injection of collagenase type IV; 24 h after surgery, MSC-derived exosomes were administered through the tail vein. The rats were euthanized at 7 or 28 days after treatment. Functional evaluation, lesion size, fiber tract integrity, axonal sprouting and white matter repair markers, biodistribution of exosomes and secretome proteomics were analyzed. DiI-labeled exosomes were found in the brains of the ICH-treated group and in the liver, lung and spleen. Animals receiving treatment with exosomes showed significantly better results in terms of functional recovery, lesion size, fiber tract integrity, axonal sprouting and white matter repair markers compared with the control group 28 days after stroke. Proteomics analysis of the exosomes identified more than 2000 proteins that could be implicated in brain repair function. In conclusion, white matter integrity was partly restored by exosome administration mediated by molecular repair factors. Exosomes as a treatment could be a heterogeneous process by nature and presents many factors that can influence brain plasticity in an adaptable and versatile manner.

White Matter Repair After Extracellular Vesicles Administration in an Experimental Animal Model of Subcortical Stroke.

Mesenchymal stem cells have previously been shown to mediate brain repair after stroke; they secrete 50-100 nm complexes called extracellular vesicles (EVs), which could be responsible for provoking neurovascular repair and functional recovery. EVs have been observed by electron microscopy and NanoSight, and they contain associated proteins such as CD81 and Alix. This purified, homogeneous population of EVs was administered intravenously after subcortical stroke in rats. To evaluate the EVs effects, we studied the biodistribution, proteomics analysis, functional evaluation, lesion size, fiber tract integrity, axonal sprouting and white matter repair markers. We found that a single administration of EVs improved functional recovery, fiber tract integrity, axonal sprouting and white matter repair markers in an experimental animal model of subcortical stroke. EVs were found in the animals' brain and peripheral organs after euthanasia. White matter integrity was in part restored by EVs administration mediated by molecular repair factors implicated in axonal sprouting, tract connectivity, remyelination and oligodendrogenesis. These findings are associated with improved functional recovery. This novel role for EVs presents a new perspective in the development of biologics for brain repair.

Stem Cell Therapy and Administration Routes After Stroke.

Cell-based therapy has demonstrated safety and efficacy in experimental animal models of stroke, as well as safety in stroke patients. However, various questions remain regarding the therapeutic window, dosage, route of administration, and the most appropriate cell type and source, as well as mechanisms of action and immune-modulation to optimize treatment based on stem cell therapy. Various delivery routes have been used in experimental stroke models, including intracerebral, intraventricular, subarachnoid, intra-arterial, intraperitoneal, intravenous, and intranasal routes. From a clinical point of view, it is necessary to demonstrate which is the most feasible, safest, and most effective for use with stroke patients. Therefore, further experimental studies concerning the safety, efficacy, and mechanisms of action involved in these therapeutic effects are required to determine their optimal clinical use.

Enhanced brain-derived neurotrophic factor delivery by ultrasound and microbubbles promotes white matter repair after stroke.

Ultrasound-targeted microbubble destruction (UTMD) has been shown to be a promising tool to deliver proteins to select body areas. This study aimed to analyze whether UTMD was able to deliver brain-derived neurotrophic factor (BDNF) to the brain, enhancing functional recovery and white matter repair, in an animal model of subcortical stroke induced by endothelin (ET)-1. UTMD was used to deliver BDNF to the brain 24 h after stroke. This technique was shown to be safe, given there were no cases of hemorrhagic transformation or blood brain barrier (BBB) leakage. UTMD treatment was associated with increased brain BDNF levels at 4 h after administration. Targeted ultrasound delivery of BDNF improved functional recovery associated with fiber tract connectivity restoration, increasing oligodendrocyte markers and remyelination compared to BDNF alone administration in an experimental animal model of white matter injury.

Adipose tissue-derived mesenchymal stem cells as a strategy to improve recovery after stroke.

INTRODUCTION: Based on the positive results observed in experimental animal models, adipose tissue-derived mesenchymal stem cells (AD-MSCs) constitute a promising therapy for stroke treatment. However, several aspects need to be clarified to identify the optimal conditions for successful clinical translation. AREAS COVERED: This review focuses on AD-MSC treatment for ischemic and hemorrhagic stroke in experimental animal models. In addition, we will explore the optimization of treatment conditions including AD-MSC production, administration routes and therapeutic windows for their appropriate use in patients. Finally we will provide an update on clinical trials on this therapy. EXPERT OPINION: Compared with other cell types, AD-MSCs have been less investigated in stroke studies. Currently, experimental animal models have shown safety and efficacy with this treatment after stroke. Due to several advantages of AD-MSCs, such as their abundance and accessibility, they can be considered a promising strategy for use in patients. However, many questions are still to be resolved regarding their mechanisms of action, immune system modulation and the effects of AD-MSCs on all components of the brain that may be affected after ischemic and hemorrhagic strokes.

Blood glutamate grabbing does not reduce the hematoma in an intracerebral hemorrhage model but it is a safe excitotoxic treatment modality.

Recent studies have shown that blood glutamate grabbing is an effective strategy to reduce the excitotoxic effect of extracellular glutamate released during ischemic brain injury. The purpose of the study was to investigate the effect of two of the most efficient blood glutamate grabbers (oxaloacetate and recombinant glutamate oxaloacetate transaminase 1: rGOT1) in a rat model of intracerebral hemorrhage (ICH). Intracerebral hemorrhage was produced by injecting collagenase into the basal ganglia. Three treatment groups were developed: a control group treated with saline, a group treated with oxaloacetate, and a final group treated with human rGOT1. Treatments were given 1 hour after hemorrhage. Hematoma volume (analyzed by magnetic resonance imaging (MRI)), neurologic deficit, and blood glutamate and GOT levels were quantified over a period of 14 days after surgery. The results observed showed that the treatments used induced a significant reduction of blood glutamate levels; however, they did not reduce the hematoma, nor did they improve the neurologic deficit. In the present experimental study, we have shown that this novel therapeutic strategy is not effective in case of ICH pathology. More importantly, these findings suggest that blood glutamate grabbers are a safe treatment modality that can be given in cases of suspected ischemic stroke without previous neuroimaging.

White matter injury restoration after stem cell administration in subcortical ischemic stroke.

INTRODUCTION: Despite its high incidence, nerve fiber (axon and myelin) damage after cerebral infarct has not yet been extensively investigated. The aim of this study was to investigate white matter repair after adipose-derived mesenchymal stem cell (ADMSC) administration in an experimental model of subcortical stroke. Furthermore, we aimed to analyze the ADMSC secretome and whether this could be implicated in this repair function. METHODS: An animal model of subcortical ischemic stroke with white matter affectation was induced in rats by injection of endothelin-1. At 24 hours, 2 × 10(6) ADMSC were administered intravenously to the treatment group. Functional evaluation, lesion size, fiber tract integrity, cell death, proliferation, white matter repair markers (Olig-2, NF, and MBP) and NogoA were all studied after sacrifice (7 days and 28 days). ADMSC migration and implantation in the brain as well as proteomics analysis and functions of the secretome were also analyzed. RESULTS: Neither ADMSC migration nor implantation to the brain was observed after ADMSC administration. In contrast, ADMSC implantation was detected in peripheral organs. The treatment group showed a smaller functional deficit, smaller lesion area, less cell death, more oligodendrocyte proliferation, more white matter connectivity and higher amounts of myelin formation. The treated animals also showed higher levels of white matter-associated markers in the injured area than the control group. Proteomics analysis of the ADMSC secretome identified 2,416 proteins, not all of them previously described to be involved in brain plasticity. CONCLUSIONS: White matter integrity in subcortical stroke is in part restored by ADMSC treatment; this is mediated by repair molecular factors implicated in axonal sprouting, remyelination and oligodendrogenesis. These findings are associated with improved functional recovery after stroke.

Effects of intravenous administration of allogenic bone marrow- and adipose tissue-derived mesenchymal stem cells on functional recovery and brain repair markers in experimental ischemic stroke.

INTRODUCTION: Stem cell therapy can promote good recovery from stroke. Several studies have demonstrated that mesenchymal stem cells (MSC) are safe and effective. However, more information regarding appropriate cell type is needed from animal model. This study was targeted at analyzing the effects in ischemic stroke of acute intravenous (i.v.) administration of allogenic bone marrow- (BM-MSC) and adipose-derived-stem cells (AD-MSC) on functional evaluation results and brain repair markers. METHODS: Allogenic MSC (2 × 106 cells) were administered intravenously 30 minutes after permanent middle cerebral artery occlusion (pMCAO) to rats. Infarct volume and cell migration and implantation were analyzed by magnetic resonance imaging (MRI) and immunohistochemistry. Function was evaluated by the Rogers and rotarod tests, and cell proliferation and cell-death were also determined. Brain repair markers were analyzed by confocal microscopy and confirmed by western blot. RESULTS: Compared to infarct group, function had significantly improved at 24 h and continued at 14 d after i.v. administration of either BM-MSC or AD-MSC. No reduction in infarct volume or any migration/implantation of cells into the damaged brain were observed. Nevertheless, cell death was reduced and cellular proliferation significantly increased in both treatment groups with respect to the infarct group. At 14 d after MSC administration vascular endothelial growth factor (VEGF), synaptophysin (SYP), oligodendrocyte (Olig-2) and neurofilament (NF) levels were significantly increased while those of glial fiibrillary acid protein (GFAP) were decreased. CONCLUSIONS: i.v. administration of allogenic MSC - whether BM-MSC or AD-MSC, in pMCAO infarct was associated with good functional recovery, and reductions in cell death as well as increases in cellular proliferation, neurogenesis, oligodendrogenesis, synaptogenesis and angiogenesis markers at 14 days post-infarct.

Adipose tissue-derived stem cells in stroke treatment: from bench to bedside.

More recently, growing interests have brought cell therapy to the forefront of promising new approaches towards efficacious treatment for stroke. Of all cell-types, adipose tissue-derived mesenchymal stem cells (AD-MSC) are considered good candidates for stroke treatment because of their abundance and easy harvesting without invasive surgery from healthy donors. A wide number of experimental studies have demonstrated the potential of AD-MSC administration for improving functional deficits and have led to the first clinical trials in stroke patients. Trophic factor release and paracrine interactions, transdifferentiation potential, and immunomodulatory effects have all been cited as the main functional mechanisms involved in AD-MSC therapy. These properties make AD-MSC therapy of special interest in fomenting the enhancement of natural brain repair mechanisms such as neurogenesis, gliagenesis, oligodendrogenesis, axonal sprouting, nerve repair, and angiogenesis. This review will focus on studies showing promising results of AD-MSC in stroke treatment.

Stem cells for brain repair and recovery after stroke.

INTRODUCTION: Stroke is a major worldwide cause of death and disability. Currently, intravenous thrombolysis and reperfusion therapies, but not the so-called neuroprotectant drugs, have been shown to be effective for acute ischemic stroke. Thus, new strategies to promote brain plasticity are necessary. Stem cell administration is an attractive future therapeutic approach. AREAS COVERED: Brain protection and repair mechanisms are activated after stroke. This article is focused on the capacity of stem cell-based therapy to enhance this postinfarct brain plasticity and recovery. Future therapeutic considerations and prospects for stroke are discussed. EXPERT OPINION: Although cell therapy is promising in stroke treatment, mechanisms of action need to be characterized in detail. Further, the different mechanisms of axonal plasticity and remodeling involucrated in brain repair, not only in the gray but also in white matter, must be investigated through noninvasive techniques, and a multidisciplinary approach is fundamental in this.

Spatial and temporal gene expression differences in core and periinfarct areas in experimental stroke: a microarray analysis.

BACKGROUND: A large number of genes are regulated to promote brain repair following stroke. The thorough analysis of this process can help identify new markers and develop therapeutic strategies. This study analyzes gene expression following experimental stroke. METHODOLOGY/PRINCIPAL FINDINGS: A microarray study of gene expression in the core, periinfarct and contralateral cortex was performed in adult Sprague-Dawley rats (n = 60) after 24 hours (acute phase) or 3 days (delayed stage) of permanent middle cerebral artery (MCA) occlusion. Independent qRT-PCR validation (n = 12) was performed for 22 of the genes. Functional data were evaluated by Ingenuity Pathway Analysis. The number of genes differentially expressed was 2,612 (24 h) and 5,717 (3 d) in the core; and 3,505 (24 h) and 1,686 (3 d) in the periinfarct area (logFC>|1|; adjP<0.05). Expression of many neurovascular unit development genes was altered at 24 h and 3 d including HES2, OLIG2, LINGO1 and NOGO-A; chemokines like CXCL1 and CXCL12, stress-response genes like HIF-1A, and trophic factors like BDNF or BMP4. Nearly half of the detected genes (43%) had not been associated with stroke previously. CONCLUSIONS: This comprehensive study of gene regulation in the core and periinfarct areas at different times following permanent MCA occlusion provides new data that can be helpful in translational research.

CDP-choline treatment induces brain plasticity markers expression in experimental animal stroke.

We investigated the effect of CDP-choline on brain plasticity markers expression in the acute phase of cerebral infarct in an experimental animal model. Male Sprague-Dawley rats were subjected to permanent middle cerebral artery occlusion (pMCAO) and treated or not with CDP-choline (500 mg/kg) daily for 14 days starting 30 min after pMCAO. Functional status was evaluated with Roger's test; lesion volume with magnetic resonance imaging (MRI) and hematoxylin and eosin staining (H&E); cell death with TUNEL; cellular proliferation with BrdU immunohistochemistry; vascular endothelial growth factor (VEGF), synaptophysin, glial fibrillary acidic protein (GFAP) and low-density lipoprotein receptor-related protein (LRP) by immunofluorescence and Western-blot techniques. CDP-choline significantly improved functional recovery and decreased lesion volume on MRI, TUNEL-positive cell number and LRP levels at 14 days. In addition, CDP-choline significantly increased BrdU, VEGF and synaptophysin values and decreased GFAP levels in the peri-infarct zone compared with the infarct group. In conclusion, our data indicate that CDP-choline improved functional recovery after permanent middle cerebral artery occlusion in association with reductions in lesion volume, cell death and LRP expression. In fact, CDP-choline increased cell proliferation, vasculogenesis and synaptophysin levels and reduced GFAP levels in the peri-infarct area of the ischemic stroke.

CDP-choline at high doses is as effective as i.v. thrombolysis in experimental animal stroke.

Use of thrombolysis in acute ischaemic stroke may be limited by a narrow benefit/risk ratio. Pharmacological inhibition of the ischaemic cascade may constitute an effective and safer approach to stroke treatment. This study compared the effects of high doses of cytidine diphosphate-choline (CDP-choline; 1000 mg/kg) with recombinant tissue plasminogen activator (rt-PA; 5 mg/kg) in an experimental animal model of embolic stroke. Fifteen rats were embolized in the right internal carotid artery with an autologous clot and were divided into three groups: (1) infarct; (2) intravenous rt-PA 5 mg/kg 30 minutes post-embolization; and (3) CDP-choline 1000 mg/kg, intraperitoneal, three doses, 30 minutes, 24 hours, and 48 hours post-embolization. Functional evaluation scores were evaluated using Rogers test, lesion volume by haematoxylin and eosin staining, cell death with transferase-mediated dUTP nick-end labelling, and plasma levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha with enzyme-linked immunosorbent assay. In this study, CDP-choline and rt-PA produced a significant reduction in brain damage considering infarct volume, cell death, and inflammatory cytokines (tumour necrosis factor-alpha and IL-6) compared with the infarct group. Additionally, CDP-choline significantly decreased infarct volume, cell death, and IL-6 levels with respect to the rt-PA group. From these results, we conclude that high-dose CDP-choline may be an effective treatment for acute ischaemic stroke even in absence of thrombolysis.