Gene expression profiling of brain endothelial cells after experimental subarachnoid haemorrhage.

Subarachnoid haemorrhage (SAH) is a type of hemorrhagic stroke that is associated with high morbidity and mortality. New effective treatments are needed to improve outcomes. The pathophysiology of SAH is complex and includes early brain injury and delayed cerebral ischemia, both of which are characterized by blood-brain barrier (BBB) impairment. We isolated brain endothelial cells (BECs) from mice subjected to SAH by injection of blood into the prechiasmatic cistern. We used gene expression profiling to identify 707 unique genes (2.8% of transcripts, 403 upregulated, 304 downregulated, 24,865 interrogated probe sets) that were significantly differentially expressed in mouse BECs after SAH. The pathway involving prostaglandin synthesis and regulation was significantly upregulated after SAH, including increased expression of the Ptgs2 gene and its corresponding COX-2 protein. Celecoxib, a selective COX-2 inhibitor, limited upregulation of Ptgs2 in BECs. In this study, we have defined the gene expression profiling of BECs after experimental SAH and provide further insight into BBB pathophysiology, which may be relevant to other neurological diseases such as traumatic brain injury, brain tumours, ischaemic stroke, multiple sclerosis, and neurodegenerative disorders.

Lithium modulates miR-1906 levels of mesenchymal stem cell-derived extracellular vesicles contributing to poststroke neuroprotection by toll-like receptor 4 regulation.

Lithium is neuroprotective in preclinical stroke models. In addition to that, poststroke neuroregeneration is stimulated upon transplantation of mesenchymal stem cells (MSCs). Preconditioning of MSCs with lithium further enhances the neuroregenerative potential of MSCs, which act by secreting extracellular vesicles (EVs). The present work analyzed whether MSC preconditioning with lithium modifies EV secretion patterns, enhancing the therapeutic potential of such derived EVs (Li-EVs) in comparison with EVs enriched from native MSCs. Indeed, Li-EVs significantly enhanced the resistance of cultured astrocytes, microglia, and neurons against hypoxic injury when compared with controls and to native EV-treated cells. Using a stroke mouse model, intravenous delivery of Li-EVs increased neurological recovery and neuroregeneration for as long as 3 months in comparison with controls and EV-treated mice, albeit the latter also showed significantly better behavioral test performance compared with controls. Preconditioning of MSCs with lithium also changed the secretion patterns for such EVs, modifying the contents of various miRNAs within these vesicles. As such, Li-EVs displayed significantly increased levels of miR-1906, which has been shown to be a new regulator of toll-like receptor 4 (TLR4) signaling. Li-EVs reduced posthypoxic and postischemic TLR4 abundance, resulting in an inhibition of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway, decreased proteasomal activity, and declined both inducible NO synthase and cyclooxygenase-2 expression, all of which culminated in reduced levels of poststroke cerebral inflammation. Conclusively, the present study demonstrates, for the first time, an enhanced therapeutic potential of Li-EVs compared with native EVs, interfering with a novel signaling pathway that yields both acute neuroprotection and enhanced neurological recovery.

Lithium enhances post-stroke blood-brain barrier integrity, activates the MAPK/ERK1/2 pathway and alters immune cell migration in mice.

Lithium induces neuroprotection against cerebral ischemia, although the underlying mechanisms remain elusive. We have previously suggested a role for lithium in calcium regulation and (extra)cerebral vessel relaxation under non-ischemic conditions. Herein, we aimed to investigate whether or not lithium contributes to post-stroke stabilization of the blood-brain barrier (BBB) in mice. Using an oxygen-glucose-deprivation (OGD) model, we first analyzed the impact of lithium treatment on endothelial cells (EC) in vitro. Indeed, such treatment of EC exposed to OGD resulted in increased cell survival as well as in enhanced expression of tight junction proteins and P-glycoprotein. Additional in vivo studies demonstrated an increased stabilization of the BBB upon lithium treatment in stroke mice, as shown by a reduced Evans blue extravasation and an elevation of tight junction protein expression. Furthermore, stabilization of the BBB as a consequence of lithium treatment was associated with an inhibition of matrix metalloproteinase-9 activity, independent of calveolin-1 regulation. In line with this, flow cytometry analysis revealed that lithium treatment led to a decreased neutrophil invasion and an increased T cell extravasation from the blood compartment towards the brain parenchyma. We finally identified the pro-survival MAPK/ERK1/2 pathway as the key regulator of the impact of lithium on the BBB. In conclusion, we demonstrate for the first time that lithium is able to enhance post-stroke BBB integrity. Importantly, our work delivers novel insights into the exact mechanism of lithium-induced acute neuroprotection, providing critical information for future clinical trials involving lithium treatment in stroke patients.

Complex Clearance Mechanisms After Intraventricular Hemorrhage and rt-PA Treatment-a Review on Clinical Trials.

Intracerebral hemorrhage in combination with intraventricular hemorrhage (IVH) is a severe type of stroke frequently leading to prolonged clinical care, continuous disability, shunt dependency, and high mortality. The molecular mechanisms induced by IVH are complex and not fully understood. Moreover, the treatment options for IVH are limited. Intraventricular recombinant tissue plasminogen activator (rt-PA) dissolves the blood clot in the ventricular system; however, whether the clinical outcome is thereby positively affected is still being debated. The mechanistic cascade induced by intraventricular rt-PA therapy may cure and harm in parallel. Despite the fact that intraventricular blood clots are thereby dissolved, blood derivatives enter the parenchyma and may still adversely affect functional structures of the brain: Smaller blood clots may obstruct the perivascular (Virchow-Robin) space and thereby the glymphatic system with detrimental consequences for cerebrospinal fluid (CSF)/interstitial fluid (ISF) flow. These clots, blood cells but also blood derivatives in the perivascular space, destabilize the blood-brain barrier from the brain parenchyma side, thereby also functionally weakening the neurovascular unit. This may lead to further accommodation of serum proteins in the ISF and particularly in the perivascular space further contributing to the adverse effects on the neuronal microenvironment. Finally, the arterial (Pacchionian) granulations have to cope with ISF containing this "blood, cell, and protein cocktail," resulting in obstruction and insufficient function of the arterial granulations, followed by a malresorptive hydrocephalus. Particularly in light of currently improved knowledge on the physiologic and pathophysiologic clearance of cerebrospinal fluid and interstitial fluid, a critical discussion and reevaluation of our current therapeutic strategies to treat intraventricular hemorrhages are needed to successfully treat patients suffering from this severe type of stroke. In this review, we therefore summarize and discuss recent clinical trials and future directions for the field of IVH with respect to the currently increased understanding of the glymphatic system and the neurovascular unit pathophysiology.

Neuronal Culture Microenvironments Determine Preferences in Bioenergetic Pathway Use.

In the brain, metabolic supply and demand is directly coupled to neuronal activation. Methods for culturing primary rodent brain cells have come of age and are geared toward sophisticated modeling of human brain physiology and pathology. However, the impact of the culture microenvironment on neuronal function is rarely considered. Therefore, we investigated the role of different neuronal culture supplements for neuronal survival and metabolic activity in a model of metabolic deprivation of neurons using oxygen deprivation, glucose deprivation, as well as live cell metabolic flux analysis. We demonstrate the impact of neuronal culture conditions on metabolic function and neuronal survival under conditions of metabolic stress. In particular, we find that the common neuronal cell culture supplement B27 protects neurons from cell death under hypoxic conditions and inhibits glycolysis. Furthermore, we present data that B27 as well as the alternative neuronal culture supplement N2 restrict neuronal glucose metabolism. On the contrary, we find that the more modern supplement GS21 promotes neuronal energy metabolism. Our data support the notion that careful control of the metabolic environment is an essential component in modeling brain function and the cellular and molecular pathophysiology of brain disease in culture.

C-reactive protein and cardiovascular risk in bipolar disorder patients: A systematic review.

OBJECTIVES: New research is revealing a strong association between inflammatory markers with bipolar disorder (BD), potentially due to the high prevalence of cardiovascular disease and cardiovascular risk factors in BD. We aimed to synthesize the literature examining the association between the clinically most relevant inflammatory marker, C-reactive protein (CRP) and cardiovascular disease and cardiovascular risk factors in patients with BD. METHODS: MEDLINE, Embase and PsychInfo were systematically searched for all relevant English language articles published prior to April 2017. Articles were included if they examined the association between CRP and cardiovascular risk factors/disease in BD. RESULTS: Fifteen relevant articles were retrieved. Studies were mostly cross-sectional and heterogeneous in the cardiovascular risk factors investigated. Overall, elevated CRP was associated with increased risk of metabolic syndrome, elevated body mass index, higher waist circumference, and obesity. CRP was inconsistently associated with elevated fasting glucose, insulin levels, serum triglycerides, total cholesterol levels, and low high density lipoprotein (HDL) levels. Atypical antipsychotic use may mediate some of these effects. No study examined CRP's association with actual cardiovascular disease (e.g. coronary artery disease) in BD. CONCLUSIONS: In BD, CRP is associated with increases in several cardiovascular risk factors, suggesting that systemic inflammation could be a shared driving force for both outcomes of BD and cardiovascular risk. Further longitudinal research is needed in this area to verify causality, including an examination of actual cardiovascular disease. Non-pharmacological and pharmacological treatments with anti-inflammatory effects should also be investigated, particularly in patients with increased CRP, for their potential to reduce cardiovascular risk in BD.

Effects of Preoperative Simulation on Minimally Invasive Hybrid Lumbar Interbody Fusion.

OBJECTIVE: The main focus of this study was to evaluate how preoperative simulation affects the surgical work flow, radiation exposure, and outcome of minimally invasive hybrid lumbar interbody fusion (MIS-HLIF). METHODS: A total of 132 patients who underwent single-level MIS-HLIF were enrolled in a cohort study design. Dose area product was analyzed in addition to surgical data. Once preoperative simulation was established, 66 cases (SIM cohort) were compared with 66 patients who had previously undergone MIS-HLIF without preoperative simulation (NO-SIM cohort). RESULTS: Dose area product was reduced considerably in the SIM cohort (320 cGy·cm2 NO-SIM cohort: 470 cGy·cm2; P < 0.01). Surgical time was shorter for the SIM cohort (155 minutes; NO-SIM cohort, 182 minutes; P < 0.05). SIM cohort had a better outcome in Numeric Rating Scale back at 6 months follow-up compared with the NO-SIM cohort (P < 0.05). CONCLUSIONS: Preoperative simulation reduced radiation exposure and resulted in less back pain at the 6 months follow-up time point. Preoperative simulation provided guidance in determining the correct cage height. Outcome controls enabled the surgeon to improve the procedure and the software algorithm.

Conditioned Medium Derived from Neural Progenitor Cells Induces Long-term Post-ischemic Neuroprotection, Sustained Neurological Recovery, Neurogenesis, and Angiogenesis.

Adult neural progenitor cells (NPCs) induce post-ischemic long-term neuroprotection and brain remodeling by releasing of survival- and plasticity-promoting mediators. To evaluate whether secreted factors may mimic neuroprotective and restorative effects of NPCs, we exposed male C57BL6 mice to focal cerebral ischemia and intravenously applied conditioned medium (CM) derived from subventricular zone NPCs. CM dose-dependently reduced infarct volume and brain leukocyte infiltration after 48 h when delivered up to 12 h after focal cerebral ischemia. Neuroprotection persisted in the post-acute stroke phase yielding enhanced neurological recovery that lasted throughout the 28-day observation period. Increased Bcl-2, phosphorylated Akt and phosphorylated STAT-3 abundance, and reduced caspase-3 activity and Bax abundance were noted in ischemic brains of CM-treated mice at 48 h post-stroke, indicative of enhanced cell survival signaling. Long-term neuroprotection was associated with increased brain glial cell line-derived neurotrophic factor (GDNF) and vascular endothelial growth factor (VEGF) concentrations at 28 days resulting in increased neurogenesis and angiogenesis. The observation that NPC-derived CM induces sustained neuroprotection and neurological recovery suggests that cell transplantation may be dispensable when secreted factors are instead administered.

Headache in Caucasian patients with Moyamoya angiopathy - a systematic cohort study.

Background Headache is common in patients with Moyanoya angiopathy (MMA), but usually underestimated in its management and not well characterized. Methods A validated self-administered headache screening questionnaire and a telephone interview were used in order to investigate headache characteristics, frequency and pain intensity in a large cohort of 55 German patients with MMA. Results Thirty-seven patients (67.3%) had suffered from headache in the past year. Headache intensity was rated 3.2 ± 1.3 on a verbal rating scale from 0 to 10. Seventeen patients (47.9%) reported migraine-like headache, 10 patients (27.0%) reported tension type-like headache and 10 patients (27.0%) had a combination of both. The majority of patients with migraine-like headache ( n = 10, 58.8%) described migrainous aura. Headache frequency and intensity improved significantly after revascularization surgery; however, nine patients developed new-onset headache postoperatively. Conclusion Headache is very common in MMA, often with a migraine-like phenotype. Tension type-like headache was also found in 27% of patients, which is a new finding that has not been reported before.

Low-Dose Lithium Stabilizes Human Endothelial Barrier by Decreasing MLC Phosphorylation and Universally Augments Cholinergic Vasorelaxation Capacity in a Direct Manner.

Lithium at serum concentrations up to 1 mmol/L has been used in patients suffering from bipolar disorder for decades and has recently been shown to reduce the risk for ischemic stroke in these patients. The risk for stroke and thromboembolism depend not only on cerebral but also on general endothelial function and health; the entire endothelium as an organ is therefore pathophysiologically relevant. Regardless, the knowledge about the direct impact of lithium on endothelial function remains poor. We conducted an experimental study using lithium as pharmacologic pretreatment for murine, porcine and human vascular endothelium. We predominantly investigated endothelial vasorelaxation capacities in addition to human basal and dynamic (thrombin-/PAR-1 receptor agonist-impaired) barrier functioning including myosin light chain (MLC) phosphorylation (MLC-P). Low-dose therapeutic lithium concentrations (0.4 mmol/L) significantly augment the cholinergic endothelium-dependent vasorelaxation capacities of cerebral and thoracic arteries, independently of central and autonomic nerve system influences. Similar concentrations of lithium (0.2-0.4 mmol/L) significantly stabilized the dynamic thrombin-induced and PAR-1 receptor agonist-induced permeability of human endothelium, while even the basal permeability appeared to be stabilized. The lithium-attenuated dynamic permeability was mediated by a reduced endothelial MLC-P known to be followed by a lessening of endothelial cell contraction and paracellular gap formation. The well-known lithium-associated inhibition of inositol monophosphatase/glycogen synthase kinase-3-ß signaling-pathways involving intracellular calcium concentrations in neurons seems to similarly occur in endothelial cells, too, but with different down-stream effects such as MLC-P reduction. This is the first study discovering low-dose lithium as a drug directly stabilizing human endothelium and ubiquitously augmenting cholinergic endothelium-mediated vasorelaxation. Our findings have translational and potentially clinical impact on cardiovascular and cerebrovascular disease associated with inflammation explaining why lithium can reduce, e.g., the risk for stroke. However, further clinical studies are warranted.

A differential impact of lithium on endothelium-dependent but not on endothelium-independent vessel relaxation.

Lithium is drug for bipolar disorders with a narrow therapeutic window. Lithium was recently reported to prevent stroke and protect vascular endothelium but tends to accumulate particularly in the brain and kidney. Here, adverse effects are common; however mechanisms are still vaguely understood. If lithium could also negatively influence the endothelium is unclear. We hypothesize that at higher lithium levels, the effects on endothelium reverses--that lithium also impairs endothelial-dependent relaxation of blood vessels. Vessel grafts from de-nerved murine aortas and porcine middle cerebral arteries were preconditioned using media supplemented with lithium chloride or acetate (0.4-100 mmol/L). Native or following phenylephrine-induced vasoconstriction, the relaxation capacity of preconditioned vessels was assessed by isometric myography, using acetylcholine to test the endothelium-dependent or sodium nitroprusside to test the endothelium-independent vasorelaxation, respectively. At the 0.4 mmol/L lithium concentration, acetylcholine-induced endothelium-dependent vessel relaxation was slightly increased, however, diminished in a concentration-dependent manner in vessel grafts preconditioned with lithium at higher therapeutic and supratherapeutic concentrations (0.8-100 mmol/L). In contrast, endothelium-independent vasorelaxation remained unaltered in preconditioned vessel grafts at any lithium concentration tested. Lithium elicits opposing effects on endothelial functions representing a differential impact on the endothelium within the narrow therapeutic window. Lithium accumulation or overdose reduces endothelium-dependent but not endothelium-independent vasorelaxation. The differentially modified endothelium-dependent vascular response represents an additional mechanism contributing to therapeutic or adverse effects of lithium.

An update on the management of post-traumatic headache.

Recent studies from the UK give the debate about how to deal best with patients suffering from whiplash injury new impetus. Following whiplash injury, about 90% of patients complain about head and/or neck pain, as well as dizziness, sleep problems and anxiety. These symptoms are often referred to as whiplash-associated disorders. In the majority of cases, these complaints develop within a few days or weeks following the accident. However, 30-50% of patients experience prolonged symptoms for more than 6 months, with headache as the main complaint. In accordance with the bio-psycho-social model of chronic post-traumatic headache, the following treatment options have been suggested: (1) proper patient education with detailed explanation of the condition; (2) support of normal movement; (3) avoiding immobilization; (4) resumption of work; and (5) targeted physiotherapy. Based on current study data, intensified physiotherapy seems not to be superior to standard therapy with simple patient education and can therefore not be recommended considering cost-benefit aspects.

Sustained neurological recovery induced by resveratrol is associated with angioneurogenesis rather than neuroprotection after focal cerebral ischemia.

According to the French paradox, red wine consumption reduces the incidence of vascular diseases even in the presence of highly saturated fatty acid intake. This phenomenon is widely attributed to the phytoalexin resveratrol, a red wine ingredient. Experimental studies suggesting that resveratrol has neuroprotective properties mostly used prophylactic delivery strategies associated with short observation periods. These studies did not allow conclusions to be made about resveratrol's therapeutic efficacy post-stroke. Herein, we systematically analyzed effects of prophylactic, acute and post-acute delivery of resveratrol (50mg/kg) on neurological recovery, tissue survival, and angioneurogenesis after focal cerebral ischemia induced by intraluminal middle cerebral artery occlusion in mice. Over an observation period of four weeks, only prolonged post-acute resveratrol delivery induced sustained neurological recovery as assessed by rota rod, tight rope and corner turn tests. Although prophylactic and acute resveratrol delivery reduced infarct volume and enhanced blood-brain-barrier integrity at 2 days post-ischemia by elevating resveratrol's downstream signal sirtuin-1, increasing cell survival signals (phosphorylated Akt, heme oxygenase-1, Bcl-2) and decreasing cell death signals (Bax, activated caspase-3), a sustained reduction of infarct size on day 28 was not observed in any of the three experimental conditions. Instead, enhanced angiogenesis and neurogenesis were noted in animals receiving post-acute resveratrol delivery, which were associated with elevated concentrations of GDNF and VEGF in the brain. Thus, sustained neurological recovery induced by resveratrol depends on successful brain remodeling rather than structural neuroprotection. The recovery promoting effect of delayed resveratrol delivery opens promising perspectives for stroke therapy.

Impurity of stem cell graft by murine embryonic fibroblasts - implications for cell-based therapy of the central nervous system.

Stem cells have been demonstrated to possess a therapeutic potential in experimental models of various central nervous system disorders, including stroke. The types of implanted cells appear to play a crucial role. Previously, groups of the stem cell network NRW implemented a feeder-based cell line within the scope of their projects, examining the implantation of stem cells after ischemic stroke and traumatic brain injury. Retrospective evaluation indicated the presence of spindle-shaped cells in several grafts implanted in injured animals, which indicated potential contamination by co-cultured feeder cells (murine embryonic fibroblasts - MEFs). Because feeder-based cell lines have been previously exposed to a justified criticism with regard to contamination by animal glycans, we aimed to evaluate the effects of stem cell/MEF co-transplantation. MEFs accounted for 5.3 ± 2.8% of all cells in the primary FACS-evaluated co-culture. Depending on the culture conditions and subsequent purification procedure, the MEF-fraction ranged from 0.9 to 9.9% of the cell suspensions in vitro. MEF survival and related formation of extracellular substances in vivo were observed after implantation into the uninjured rat brain. Impurity of the stem cell graft by MEFs interferes with translational strategies, which represents a threat to the potential recipient and may affect the graft microenvironment. The implications of these findings are critically discussed.

Gadolinium enhancement in newly diagnosed patients with lumbar disc herniations are associated with inflammatory peridiscal tissue reactions--evidence of fragment degradation?

OBJECTIVE: It is debatable whether a local inflammatory tissue response caused by herniated disc material contributes to sciatic pain and/or sensorimotor deficits. The impact of inflammatory changes on local tissue remodelling, the healing process and the clinical course of disease remains unclear. METHODS: In this prospective observational study, we included a total of 31 patients with a single-level, unilateral lumbar disc herniation. The diagnosis was confirmed by magnetic resonance imaging (MRI)±gadolinium. The presence of peridiscal contrast enhancement was correlated with the extent of inflammatory reactions in the herniated fragments as confirmed by immunohistochemistry; clinical symptoms, including the duration of radicular pain; and the incidence of sensorimotor deficits. RESULTS: Peridiscal contrast enhancement was found in 17 patients (55%) and was encasing the adjacent rootlet in 4 cases. There was no significant correlation between gadolinium uptake and the presence of sensorimotor deficits or the duration of radicular symptoms. Degenerative changes were observed in all 31 disc specimens. Overall, 18 cases exhibited increased cellularity in the marginal areas, which were mostly populated by CD68(+) macrophages and fibroblasts. Additionally, these areas displayed a limited number of CD3(+) T-lymphocytes and different degrees of concomitant neovascularisation, which represented a chronic and unspecific immune response. Peridiscal contrast enhancement on MRI was significantly correlated with the histopathological characteristics of tissue inflammation. However, no correlation was found between the histological evidence and the degree of inflammation and neurological symptoms. CONCLUSION: Gadolinium-enhanced MRI is a sensitive method to detect unspecific inflammatory reactions in therapy-naïve disc herniations. However, the neuroradiological and histological evidence of peridiscal inflammation was not correlated with the severity of pain or sensorimotor deficits in our patients. Additional research is needed because the occurrence of local inflammation may indicate an ongoing degradation of herniated fragments and thus be helpful in therapeutic decision-making.

Lithium prevents early cytosolic calcium increase and secondary injurious calcium overload in glycolytically inhibited endothelial cells.

Cytosolic free calcium concentration ([Ca(2+)]i) is a central signalling element for the maintenance of endothelial barrier function. Under physiological conditions, it is controlled within narrow limits. Metabolic inhibition during ischemia/reperfusion, however, induces [Ca(2+)]i overload, which results in barrier failure. In a model of cultured porcine aortic endothelial monolayers (EC), we addressed the question of whether [Ca(2+)]i overload can be prevented by lithium treatment. [Ca(2+)]i and ATP were analysed using Fura-2 and HPLC, respectively. The combined inhibition of glycolytic and mitochondrial ATP synthesis by 2-desoxy-d-glucose (5mM; 2-DG) plus sodium cyanide (5mM; NaCN) caused a significant decrease in cellular ATP content (14±1 nmol/mg protein vs. 18±1 nmol/mg protein in the control, n=6 culture dishes, P<0.05), an increase in [Ca(2+)]i (278±24 nM vs. 71±2 nM in the control, n=60 cells, P<0.05), and the formation of gaps between adjacent EC. These observations indicate that there is impaired barrier function at an early state of metabolic inhibition. Glycolytic inhibition alone by 10mM 2-DG led to a similar decrease in ATP content (14±2 nmol/mg vs. 18±1 nmol/mg in the control, P<0.05) with a delay of 5 min. The [Ca(2+)]i response of EC was biphasic with a peak after 1 min (183±6 nM vs. 71±1 nM, n=60 cells, P<0.05) followed by a sustained increase in [Ca(2+)]i. A 24-h pre-treatment with 10mM of lithium chloride before the inhibition of ATP synthesis abolished both phases of the 2-DG-induced [Ca(2+)]i increase. This effect was not observed when lithium chloride was added simultaneously with 2-DG. We conclude that lithium chloride abolishes the injurious [Ca(2+)]i overload in EC and that this most likely occurs by preventing inositol 3-phosphate-sensitive Ca(2+)-release from the endoplasmic reticulum. Though further research is needed, these findings provide a novel option for therapeutic strategies to protect the endothelium against imminent barrier failure.

Pitfalls and fallacies interfering with correct identification of embryonic stem cells implanted into the brain after experimental traumatic injury.

Cell-therapy was proposed to be a promising tool in case of death or impairment of specific cell types. Correct identification of implanted cells became crucial when evaluating the success of transplantation therapy. Various methods of cell labeling have been employed in previously published studies. The use of intrinsic signaling of green fluorescent protein (GFP) has led to a well known controversy in the field of cardiovascular research. We encountered similar methodological pitfalls after transplantation of GFP-transfected embryonic stem cells into rat brains following traumatic brain injury (TBI). As the identification of implanted graft by intrinsic autofluorescence failed, anti-GFP labeling coupled to fluorescent and conventional antibodies was needed to visualize the implanted cells. Furthermore, different cell types with strong intrinsic autofluorescence were found at the sites of injury and transplantation, thus mimicking the implanted stem cells. GFP-positive stem cells were correctly localized, using advanced histological techniques. The activation of microglia/macrophages, accompanying the transplantation post TBI, was shown to be a significant source of artefacts, interfering with correct identification of implanted stem cells. Dependent on the strategy of stem cell tracking, the phagocytosis of implanted cells as observed in this study, might also impede the interpretation of results. Critical appraisal of previously published data as well as a review of different histological techniques provide tools for a more accurate identification of transplanted stem cells.

Occurrence and recurrence of spontaneous chronic subdural haematoma is associated with a factor XIII deficiency.

OBJECTIVE: In some patients, chronic subdural haematoma (cSDH) appears to occur spontaneously with frequent re-bleeding events. The pathophysiology of this phenomenon is still poorly understood. Because coagulation factor XIII (FXIII) is known to be involved in vascular integrity, endothelial barrier function and wound healing, we evaluated the role of FXIII in spontaneous cSDH. METHODS: We prospectively scrutinised the origin of cSDH in 117 patients and identified a subgroup of patients suffering from spontaneous cSDH who were included in this study. We analysed the plasma activity of FXIII and standard coagulation parameters and compared these data to age- and sex-matched healthy controls. We assessed the occurrence of re-bleeding events using clinical and imaging data and compared FXIII activity in patients with and without re-bleeding events. RESULTS: Out of 117 cSDH patients, 18 individuals suffered from spontaneous cSDH in this study. The patients with spontaneous cSDH showed significantly lower FXIII activity than the control group (65% [52.75, 80.25] (median [IQR]) vs. 93% [81, 111], P=0.001), whereas standard coagulation parameters did not differ significantly between the groups. Six patients developed re-bleeding events after haematoma evacuation, and these patients expressed significantly lower FXIII activity compared to the other 12 patients (47.5% [33.5, 64] vs. 78.5% [58, 87], P=0.005). The patient group with FXIII≤68.5% differed significantly from the group with FXIII>68.5% when categorised by the occurrence of re-bleeding events (n=6/9 vs. n=0/9, P=0.009). This cut-off value predicted the re-bleeding events with a sensitivity of 100% and a specificity of 75% (positive predictive value: 66%, negative predictive value: 100%). CONCLUSION: FXIII deficiency may play a pathophysiological role in spontaneous cSDH, so we suggest investigating FXIII activity because it may predict re-bleeding events after treatment. In individuals with considerably low FXIII activity, FXIII substitution may mitigate the chronic nature of this disease.