Neuroinflammation contributes to the pathophysiology of diverse diseases including stroke, traumatic brain injury, Alzheimer's Disease, Parkinson's Disease, and multiple sclerosis, resulting in neurodegeneration and loss of neurological function. The response of the microvascular endothelium often contributes to neuroinflammation. One such response is the up-regulation of endothelial adhesion molecules which facilitate neutrophil adhesion to the endothelium and their migration from blood to tissue. Neuregulin-1 (NRG1) is an endogenous growth factor which has been reported to have anti-inflammatory effects in experimental stroke models. We hypothesized that NRG1 would decrease the endothelial response to inflammation, and result in a decrease in neutrophil adhesion to endothelial cells. We tested this hypothesis in an in-vitro model of cytokine-induced endothelial injury, in which human brain microvascular endothelial cells (BMECs) were treated with IL-1β, along with co-incubation with vehicle or NRG1-β. Outcome measures included protein levels of endothelial ICAM-1, VCAM-1, and E-selectin; as well as the number of neutrophils that adhere to the endothelial monolayer. Our data show that NRG1-β decreased the levels of VCAM-1, E-selectin, and neutrophil adhesion to brain microvascular endothelial cells activated by IL1-β. These findings open new possibilities for investigating NRG1 in neuroprotective strategies in brain injury.
neuroinflammation; IL-1β; NRG1-β; neutrophil; VCAM-1; E-selectin
Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited form of cerebral small vessel disease caused by mutations in conserved residues of NOTCH3. Affected arteries of CADASIL feature fibrosis and accumulation of NOTCH3. A variety of collagen subtypes (types I, III, IV, and VI) have been identified in fibrotic CADASIL vessels. Biglycan (BGN) and decorin (DCN), are Class I members of the small leucine-rich proteoglycan (SLRP) family that regulate collagen fibril size. Because DCN has been shown to deposit in arteries in cerebral small vessel disease, we tested whether BGN accumulates in arteries of CADASIL brains. BGN was strongly expressed in both small penetrating and leptomeningeal arteries of CADASIL brain. BGN protein was localized to all three layers of arteries (intima, media, and adventitia). Substantially more immunoreactivity was observed in CADASIL brains compared to controls. Immunoblotting of brain lysates showed a 4-fold increase in CADASIL brains (compared to controls). Messenger RNA encoding BGN was also increased in CADASIL and was localized by in situ hybridization to all three vascular layers in CADASIL. Human cerebrovascular smooth muscle cells exposed to purified NOTCH3 ectodomain upregulated BGN, DCN, and COL4A1 through mechanisms that are sensitive to rapamycin, a potent mTOR inhibitor. In addition, BGN protein interacted directly with NOTCH3 protein in cell culture and in direct protein interaction assays. In conclusion, BGN is a CADASIL-enriched protein that potentially accumulates in vessels by mTOR-mediated transcriptional activation and/or post-translational accumulation via protein interactions with NOTCH3 and collagen.
CADASIL; small leucine rich proteoglycans; biglycan; collagen; Notch; arteries; protein interactions
Intracerebral hemorrhage; Perihematoma edema; Secondary injury
bioassay; blood clot; blood flow; embolic stroke; IDE; IND; NDA; neuroprotection; thrombolytic; safety; toxicity
Stroke is the fourth leading cause of death. Despite decades of research, no neuroprotective drug has proven to be effective clinically. One widely accepted view to account for this negative outcome is that the rodent stroke model simply does not adequately reflect the complexity of human stroke. Recent failures of several high-profile neuroprotective drugs for stroke treatment in phase III clinical trials further underscore the importance of developing adequate animal models for stroke research. The brain organization and vascular circuitry of nonhuman primates (NHPs) are more homologous with humans than the widely used rodent for stroke modeling. The Stroke Therapy Academic Industry Roundtable, a national committee commissioned by the American Heart Association, recommended that clinically relevant NHP stroke models be established for developing and assessing neuroprotective drugs. The aim of this article is to review the challenges and applications of magnetic resonance imaging studies of NHP stroke models.
Nonhuman primate; MRI; Perfusion; Diffusion; Functional MRI; MCAO
Recombinant T-cell Receptor Ligand 1000 (RTL1000), a partial human major histocompatibility complex (MHC) molecule coupled to a human myelin peptide, reduces infarct size after experimental stroke in HLA-DRB1*1502 transgenic (DR2-Tg) mice. In this study, we characterized the therapeutic time window of opportunity for RTL1000; we explored the efficacy of single dose of RTL1000 administration and determined if RTL1000 affordslong-term neurobehavioral functional improvement after ischemic stroke. Male DR2-Tg mice underwent 60 min of intraluminal reversible middle cerebral artery occlusion (MCAO). RTL1000 or vehicle was injected 4, 6 or 8 h after MCAO, followed by 3 daily injections. In single dose study, one-time injection of RTL1000 was applied 4 h after MCAO. Cortical, striatal and hemispheric infarct sizes were measured 24 h or 96 h after stroke. Behavioral testing, including neuroscore evaluation, open field, paw preference and novel object recognition was performed up to 28 days after stroke. Our data showed RTL1000 significantly reduced infarct size 96 h after MCAO when first injection was given 4 and 6, but not 8 h after the onset of stroke. A single dose of 400 µg or 100 µg RTL1000 also significantly reduced infarct size 24 h after MCAO. Behavioral testing showed RTL1000 treatment used 4 h after MCAO improved long-term cognitive outcome 28 days after stroke. Taken together, RTL1000 protects against acute injury if applied within a 6-h time window and improves long-term functional recovery after experimental stroke in DR2-Tg mice.
Ischemicstroke; Neurobehavioral evaluation; Immunotherapy; Recombinant T-cell receptor Ligand; HLA-DR2transgenic mice
Plasmin, a direct fibrinolytic, shows a significantly superior hemostatic safety profile compared to recombinant tissue plasminogen activator (rtPA), the only FDA approved thrombolytic for the treatment of acute ischemic stroke. The improved safety of plasmin is attributed to the rapid inhibition of free plasmin by endogenous plasmin inhibitors present in very high concentrations (1 μM). However, this rapid inhibition prevents the intra-venous (IV) administration of plasmin. In emergency situations catheter-based local administration is not practical. There is a need for an alternative technique for IV administration of plasmin. A possible solution is the encapsulation of plasmin in echogenic liposomes (ELIP) for protection from inhibitors until ultrasound (US)-triggered release at the clot-site. ELIP are bilayer phospholipid vesicles with encapsulated gas microbubbles. US induces oscillation and collapse of the gas bubbles, which facilitates ELIP rupture and delivery of the encapsulated contents. Plasmin-loaded ELIP (PELIP) were manufactured, and characterized for size, gas- and drug-encapsulation, and in-vitro thrombolytic efficacy using a human whole blood clot model. Clots were exposed to PELIP with and without exposure to US (center frequency 120 kHz, pulse repetition frequency 1667 Hz, peak-to-peak pressure of 0.35 MPa, 50% duty cycle). Thrombolytic efficacy was calculated by measuring the change in clot width over a 30-minute treatment period using an edge-detection MATLAB program. The mean clot lysis obtained with PELIP in the presence of US exposure was 31% higher than that obtained without US exposure, and 15% higher than that obtained with rtPA treatment (p<0.05). The enhanced clot lysis is attributed to the US-mediated release of plasmin from the liposomes.
Plasmin; Acute Ischemic Stroke; Thrombolysis; Echogenic Liposomes; Ultrasound
Collateral circulation, defined as the supplementary vascular network that maintains cerebral blood flow (CBF) when the main vessels fail, constitutes one important defense mechanism of the brain against ischemic stroke. In the present study, continuous arterial spin labeling (CASL) was used to quantify CBF and obtain perfusion territory maps of the major cerebral arteries in spontaneously hypertensive rats (SHR) and their normotensive Wistar-Kyoto (WKY) controls. Results show that both WKY and SHR have complementary, yet significantly asymmetric perfusion territories. Right or left dominances were observed in territories of the anterior (ACA), middle and posterior cerebral arteries, and the thalamic artery. Magnetic resonance angiography showed that some of the asymmetries were correlated with variations of the ACA. The leptomeningeal circulation perfusing the outer layers of the cortex was observed as well. Significant and permanent changes in perfusion territories were obtained after temporary occlusion of the right middle cerebral artery in both SHR and WKY, regardless of their particular dominance. However, animals with right dominance presented a larger volume change of the left perfusion territory (23 ± 9%) than animals with left dominance (7 ± 5%, P < 0.002). The data suggest that animals with contralesional dominance primarily safeguard local CBF values with small changes in contralesional perfusion territory, while animals with ipsilesional dominance show a reversal of dominance and a substantial increase in contralesional perfusion territory. These findings show the usefulness of CASL to probe the collateral circulation.
cerebral blood flow; collateral flow; magnetic resonance imaging; vascular territory
Astrocyte swelling (cytotoxic brain edema) is the major neurological complication of acute liver failure (ALF), a condition in which ammonia has been strongly implicated in its etiology. Ion channels and transporters are known to be involved in cell volume regulation and a disturbance in these systems may result in cell swelling. One ion channel known to contribute to astrocyte swelling/brain edema in other neurological disorders is the ATP-dependent, non-selective cation channel (NCCa-ATP channel). We therefore examined its potential role in the astrocyte swelling/brain edema associated with ALF. Cultured astrocytes treated with 5 mM ammonia showed a 3-fold increase in the sulfonylurea receptor type 1 (SUR1) protein expression, a marker of NCCa-ATP channel activity. Blocking SUR1 with glibenclamide significantly reduced the ammonia-induced cell swelling in cultured astrocytes. Additionally, overexpression of SUR1 in ammonia-treated cultured astrocytes was significantly reduced by co-treatment of cells with BAY 11-7082, an inhibitor of NF-κB, indicating the involvement of an NF-κB-mediated SUR1 upregulation in the mechanism of ammonia-induced astrocyte swelling. Brain SUR1 mRNA level was also found to be increased in the thioacetamide (TAA) rat model of ALF. Additionally, we found a significant increase in SUR1 protein expression in rat brain cortical astrocytes in TAA-treated rats. Treatment with glibenclamide significantly reduced the brain edema in this model of ALF. These findings strongly suggest the involvement of NCCa-ATP channel in the astrocyte swelling/brain edema in ALF, and that targeting this channel may represent a useful approach for the treatment of the brain edema associated with ALF.
Ammonia; acute liver failure; astrocyte swelling; brain edema; glibenclamide; NCCa-ATP channel; sulfonylurea receptor type 1 protein
Acute ischemic stroke, the most frequent cause of permanent disability in adults worldwide, results from transient or permanent reduction in regional cerebral blood flow and involves oxidative stress and inflammation. Despite the success of experimental animal models of stroke in identifying anti-inflammatory/neuroprotective compounds, translation of these putative neuroprotectants to human clinical trials has failed to produce a positive outcome. Tissue injury and stress activate endogenous mechanisms which function to restore homeostatic balance and prevent further damage by upregulating the synthesis of lipid signaling molecules, including N-palmitoylethanolamine (PEA or palmitoylethanolamide). PEA exerts neuroprotection and reduces inflammatory secondary events associated with brain ischemia reperfusion injury (middle cerebral artery occlusion (MCAo)). Here, we examined the neuroprotective potential of a co-ultramicronized composite containing PEA and the antioxidant flavonoid luteolin (10:1 by mass), nominated co-ultraPEALut. The study consisted of two arms. In the first, rats subjected to MCAo and treated with co-ultraPEALut post-ischemia showed reduced edema and brain infract volume, improved neurobehavioral functions, and reduced expression of pro-inflammatory markers and astrocyte markers. In the second arm, a cohort of 250 stroke patients undergoing neurorehabilitation on either an inpatient or outpatient basis were treated for 60 days with a pharmaceutical preparation of co-ultraPEALut (Glialia®). At baseline and after 30 days of treatment, all patients underwent a battery of evaluations to assess neurological status, impairment of cognitive abilities, the degree of spasticity, pain, and independence in daily living activities. All indices showed statistically significant gains at study end. Despite its observational nature, this represents the first description of co-ultraPEALut administration to human stroke patients and clinical improvement not otherwise expected from spontaneous recovery. Further, controlled trials are warranted to confirm the utility of co-ultraPEALut to improve clinical outcome in human stroke.
Palmitoylethanolamide; Luteolin; Co-ultramicronization; Neuroinflammation; Neuroprotection; Brain ischemia reperfusion injury
Experimental animal models of aneurysmal subarachnoid hemorrhage (SAH) have provided a wealth of information on the mechanisms of brain injury. The Rat endovascular perforation model (EVP) replicates the early pathophysiology of SAH and hence is frequently used to study early brain injury following SAH.
This paper presents a brief review of historical development of the EVP model, details the technique used to create SAH and considerations necessary to overcome technical challenges.
Previous studies show that circulating endothelial progenitor cells (EPCs) promote angiogenesis, which is a process associated with improved recovery in animal models of traumatic brain injury (TBI), and that recombinant human erythropoietin (rhEPO) plays a protective role following stroke. Thus, it was hypothesized that rhEPO would enhance recovery following brain injury in a rat model of TBI via an increase in the mobilization of EPCs and, subsequently, in angiogenesis. Flow cytometry assays using CD34− and CD133-specific antibodies were utilized to identify alterations in EPC levels, CD31 and CD34 antibody-stained brain tissue sections were used to quantify angiogenesis, and the Morris water maze (MWM) test and the modified Neurological Severity Score (mNSS) test were used to evaluate behavioral recovery. Compared with saline treatment, treatment with rhEPO significantly increased the number of circulating EPCs on days 1, 4, 7, and 14 (P<0.05), improved spatial learning ability on days 24 and 25 (P<0.05), and enhanced memory recovery on day 26 (P<0.05). Moreover, rhEPO treatment decreased mNSS assessment scores on days 14, 21, and 25 (P<0.05). There was a strong correlation between levels of circulating EPCs and CD34− and CD31-positive cells within the injured boundary zone (CD34+
r=0.910, P<0.01; CD31+
r=0.894, P<0.01) and the ipsilateral hippocampus (CD34+
r=0.841, P<0.01; CD31+
r=0.835, P<0.01). The present data demonstrate that rhEPO treatment improved functional outcomes in rats following TBI via an increase in the mobilization of EPCs and in subsequent angiogenesis.
Traumatic brain injury; Erythropoietin; Endothelial progenitor cells; Angiogenesis
A balanced immune system response plays an important role in acute ischemic stroke (AIS) recovery. Our laboratory has previously identified several immune-related genes, including arginase 1 (ARG1), with altered expression in human AIS patients. The neutrophil-lymphocyte ratio (NLR) may be a marker of the degree of immune dysregulation following AIS; however, the molecular mechanisms that may mediate the NLR are unknown. The purpose of this study was to (1) examine the relationship between ARG1, NLR, and AIS severity and (2) to utilize principal component analysis (PCA) to statistically model multiple gene expression changes following AIS. AIS patients and stroke-free control subjects were recruited, and blood samples were collected from AIS patients within 24 h of stroke symptom onset. White blood cell differentials were obtained at this time to calculate the NLR. Gene expression was measured using real-time PCR. PCA with varimax rotation was used to develop composite variables consisting of a five-gene profile. ARG1 was positively correlated with NLR (r = 0.57, p = 0.003), neutrophil count (r = 0.526, p = 0.007), NIHSS (r = 0.607, p = 0.001), and infarct volume (r = 0.27, p = 0.051). PCA identified three principal components that explain 84.4 % of variation in the original patient gene dataset comprised of ARG1, LY96, MMP9, s100a12, and PC1 was a significant explanatory variable for NIHSS (p < 0.001) and NLR (p = 0.005). Our study suggests a novel relationship between ARG1, NLR, and stroke severity, and the NLR is an underutilized clinically available biomarker to monitor the post-stroke immune response.
Stroke; Immune system; Arginase; Neutrophil-lymphocyte ratio
Brain arteriovenous malformations (AVMs) are abnormal connections of arteries and veins, resulting in arteriovenous shunting of blood. Primary medical therapy is lacking; treatment options include surgery, radiosurgery, and embolization, often in combination. Judicious selection of AVM patients for treatment requires balancing risk of treatment complications against the risk of hemorrhage in the natural history course. This review focuses on the epidemiology, hemorrhage risk, and factors influencing risk of hemorrhage in the untreated natural course associated with sporadic brain AVM.
Arteriovenous malformation; Epidemiology; Intracerebral hemorrhage; Natural history; Risk factor; Survival; Treatment
Despite extensive research into stroke pathology, there have not been any major recent advancements in stroke therapeutics. Animal models of cerebral ischemia and clinical data have been used to investigate the progressive neural injury that occurs after an initial ischemic insult. This has lead researchers to focus more on the peripheral immune response that is generated as a result of cerebral ischemia. The therapies that have been developed as a result of this research thus far have proven ineffective in clinical trials. The failure of these therapeutics in clinical trials is thought to be due to the broad immunosuppression elicited as a result of the treatments and the cerebral ischemia itself. Emerging evidence indicates a more selective modulation of the immune system following stroke could be beneficial. The spleen has been shown to exacerbate neural injury following experimental stroke and would provide a strong therapeutic target. Selecting facets of the immune system to target would allow the protective and regenerative properties of the immune response to remain intact while blunting the pro-inflammatory response generated towards the injured brain.
spleen; interferon gamma; stroke; T cells
Acute post-traumatic ventricular dilation and hydrocephalus are relatively frequent consequences of traumatic brain injury (TBI). Several recent studies have indicated that high iron level in brain may relate to hydrocephalus development after intracranial hemorrhage. However, the role of iron in the development of post-traumatic hydrocephalus is still unclear. This study was to determine whether or not iron has a role in hydrocephalus development after TBI. TBI was induced by lateral fluid-percussion in male Sprague-Dawley rats. Some rats had intraventricular injection of iron. Acute hydrocephalus was measured by magnetic resonance T2-weighted imaging and brain hemorrhage was determined by T2* gradient-echo sequence imaging and brain hemoglobin levels. The effect of deferoxamine on TBI-induced hydrocephalus was examined. TBI resulted in acute hydrocephalus at 24 hours (lateral ventricle volume: 24.1±3.0 vs. 9.9±0.2 mm3 in sham group). Intraventricular injection of iron also caused hydrocephalus (25.7 ± 3.4 vs. 9.0 ± 0.6 mm3 in saline group). Deferoxamine treatment attenuated TBI-induced hydrocephalus and heme oxygenase-1 upregulation. In conclusion, iron may contribute to acute hydrocephalus after TBI.
Deferoxamine; hydrocephalus; lateral fluid percussion; traumatic brain injury
RTL1000 is a partial human MHC molecule coupled to a human myelin peptide. We previously demonstrated that RTL1000 was protective against experimental ischemic stroke in HLA-DR2 transgenic (DR2-Tg) mice. Since thrombolysis with recombinant tissue plasminogen activator (t-PA) is a standard therapy for stroke, we determined if RTL1000 efficacy is altered when combined with t-PA in experimental stroke. Male DR2-Tg mice underwent 60 min of intraluminal middle cerebral artery occlusion (MCAO). t-PA or vehicle was infused intravenously followed by either a single or 4 daily subcutaneous injections of RTL1000 or vehicle. Infarct size was measured by 2, 3, 5-triphenyltetrazolium chloride staining at 24h or 96 h of reperfusion. Our data showed that t-PA alone reduced infarct size when measured at 24 h but not at 96 h after MCAO. RTL1000 alone reduced infarct size both at 24 and 96h after MCAO. Combining RTL1000 with t-PA did not alter its ability to reduce infarct size at either 24 or 96 h after MCAO and provides additional protection in t-PA treated mice at 24 h after ischemic stroke. Taken together, RTL1000 treatment alone improves outcome and provides additional protection in t-PA treated mice in experimental ischemic stroke.
Ischemic stroke; Immunotherapy; Recombinant T-cell receptor Ligand; tissue plasminogen activator; HLA-DR2 transgenic mice
This perspective article uses a new concept named vascular neural network as an umbrella to redefine vascular pathophysiology for subarachnoid hemorrhage (SAH) induced vasospasm and early/delayed brain injury. Five vascular components are discussed including large artery moderate vasospasm which may not contribute to reduced cerebral blood flow (CBF) and poor outcomes after SAH. Even severe vasospasm alone with lumen diameter narrowing less than 75% of the normal diameter may not cause delayed brain injury, unless it is combined with peripheral and distal smaller artery dysfunctions. Vasospasm in smaller artery or arterioles contributes to the reduction of CBF and poor outcomes after SAH, because of limited or no collateral circulation reserves. Capillary or pre-capillary pear-string-type of contraction may block red blood cell flow and astrocyte edema compression may contribute to the loss of capillary density after SAH. Venules may be compressed by brain edema because venules have a thin wall of only one layer of endothelial cells with adventitia tissues. Deep cerebral vein vasospasm reduces venous flow and may cause venous infarction. When venous flow is obstructed, it is presumed that arterial dilatation may enhance brain edema and be harmful. Overall, all of these five vascular components in the vascular neural network are interrelated and more than one component or even all five components may be affected after SAH. All of these vascular components should be taken into consideration for patient care. Studying potential roles of venules and deep veins in the outcome of SAH patients and mechanisms of venule compression and vein spasm may be new aspects for future investigations.
vasospasm; subarachnoid hemorrhage; vascular neural network
Thrombin causes blood-brain barrier disruption and this study examined whether thrombin can cause brain hemorrhage through protease-activated receptor-1 (PAR-1). Male wild type and PAR-1 knockout mice had an intracerebral injection of thrombin or saline. Mice then underwent serial T2 magnetic resonance imaging and were euthanized for brain hemoglobin, iron and interleukin-1β measurements. Thrombin caused massive T2 lesions and brain hemorrhage in wild type mice. These effects were markedly reduced in PAR-1 knockout mice. Thrombin also increased brain interleukin-1β and this was absent in PAR-1 knockout mice. In conclusion, thrombin increases interleukin-1β levels and induces intracerebral hemorrhage through PAR-1 activation.
cerebral hemorrhage; interleukin-1β; protease activated receptor-1(PAR-1); thrombin
The classical neurovascular unit (NVU), composed primarily of endothelium, astrocytes and neurons, could be expanded to include smooth muscle and perivascular nerves present in both the up and down stream feeding blood vessels (arteries and veins). The extended NVU, which can be defined as the vascular neural network (VNN), may represent a new physiological unit to consider for therapeutic development in stroke, traumatic brain injury, and other brain disorders . This review is focused on traumatic brain injury and resultant post-traumatic changes in cerebral blood-flow, smooth muscle cells, matrix, BBB structures and function and the association of these changes with cognitive outcomes as described in clinical and experimental reports. We suggest that studies characterizing TBI outcomes should increase their focus on changes to the VNN as this may yield meaningful therapeutic targets to resolve post-traumatic dysfunction.
Traumatic brain injury; juvenile traumatic injury; blood-brain barrier; neurovascular unit; cerebral blood flow; smooth muscle cells; matrix
Patients harboring brain arteriovenous malformation (bAVM) are at life-threatening risk of rupture and intracranial hemorrhage (ICH). The pathogenesis of bAVM has not been completely understood. Current treatment options are invasive and ≈ 20% of patients are not offered interventional therapy because of excessive treatment risk. There are no specific medical therapies to treat bAVMs. The lack of validated animal models has been an obstacle for testing hypotheses of bAVM pathogenesis and testing new therapies. In this review, we summarize bAVM model development; and bAVM pathogenesis and potential therapeutic targets that have been identified during model development.
Activin-like kinase 1; Angiogenesis; Brain arteriovenous malformation; Conditional knockout; Endoglin; Hereditary hemorrhagic telangiectasia; Mouse models