Intrastriatal Injection of Autologous Blood or Clostridial Collagenase as Murine Models of Intracerebral Hemorrhage

Intracerebral hemorrhage (ICH) is a common form of cerebrovascular disease and is associated with significant morbidity and mortality. Lack of effective treatment and failure of large clinical trials aimed at hemostasis and clot removal demonstrate the need for further mechanism-driven investigation of ICH. This research may be performed through the framework provided by preclinical models. Two murine models in popular use include intrastriatal (basal ganglia) injection of either autologous whole blood or clostridial collagenase. Since, each model represents distinctly different pathophysiological features related to ICH, use of a particular model may be selected based on what aspect of the disease is to be studied. For example, autologous blood injection most accurately represents the brain''s response to the presence of intraparenchymal blood, and may most closely replicate lobar hemorrhage. Clostridial collagenase injection most accurately represents the small vessel rupture and hematoma evolution characteristic of deep hemorrhages. Thus, each model results in different hematoma formation, neuroinflammatory response, cerebral edema development, and neurobehavioral outcomes. Robustness of a purported therapeutic intervention can be best assessed using both models. In this protocol, induction of ICH using both models, immediate post-operative demonstration of injury, and early post-operative care techniques are demonstrated. Both models result in reproducible injuries, hematoma volumes, and neurobehavioral deficits. Because of the heterogeneity of human ICH, multiple preclinical models are needed to thoroughly explore pathophysiologic mechanisms and test potential therapeutic strategies.     

Targeted insult to subsurface cortical blood vessels using ultrashort laser pulses: three models of stroke

We present a method to produce vascular disruptions within rat brain parenchyma that targets single microvessels. We used two-photon microscopy to image vascular architecture, to select a vessel for injury and to measure blood-flow dynamics. We irradiated the vessel with high-fluence, ultrashort laser pulses and achieved three forms of vascular insult. (i) Vessel rupture was induced at the highest optical energies; this provides a model for hemorrhage. (ii) Extravasation of blood components was induced near the lowest energies and was accompanied by maintained flow in the target vessel. (iii) An intravascular clot evolved when an extravasated vessel was further irradiated. Such clots dramatically impaired blood flow in downstream vessels, in which speeds dropped to as low as approximately 10% of baseline values. This demonstrates that a single blockage to a microvessel can lead to local cortical ischemia. Lastly, we show that hemodilution leads to a restoration of flow in secondary downstream vessels.     

A mouse model of intracerebral hemorrhage using autologous blood infusion

The development of controllable and reproducible animal models of intracerebral hemorrhage (ICH) is essential for the systematic study of the pathophysiology and treatment of hemorrhagic stroke. In recent years, we have used a modified version of a murine ICH model to inject blood into mouse basal ganglia. According to our protocol, autologous blood is stereotactically infused in two stages into the right striatum to mimic the natural events of hemorrhagic stroke. Following ICH induction, animals demonstrate reproducible hematomas, brain edema formation and marked neurological deficits. Our technique has proven to be a reliable and reproducible means of creating ICH in mice in a number of acute and chronic studies. We believe that our model will serve as an ideal paradigm for investigating the complex pathophysiology of hemorrhagic stroke. The protocol for establishing this model takes about 2 h.     

Dynamics of pruning in simulated large-scale spiking neural networks

Massive synaptic pruning following over-growth is a general feature of mammalian brain maturation. This article studies the synaptic pruning that occurs in large networks of simulated spiking neurons in the absence of specific input patterns of activity. The evolution of connections between neurons were governed by an original bioinspired spike-timing-dependent synaptic plasticity (STDP) modification rule which included a slow decay term. The network reached a steady state with a bimodal distribution of the synaptic weights that were either incremented to the maximum value or decremented to the lowest value. After 1x10(6) time steps the final number of synapses that remained active was below 10% of the number of initially active synapses independently of network size. The synaptic modification rule did not introduce spurious biases in the geometrical distribution of the remaining active projections. The results show that, under certain conditions, the model is capable of generating spontaneously emergent cell assemblies.     

Recurrent hemorrhage from corpus luteum during anticoagulant therapy.

A 43-year old woman had recurrent massive intraperitoneal hemorrhage from rupture of a hemorrhagic corpus luteum in two successive menstrual cycles while receiving anticoagulant therapy. Left oophorectomy was performed on the first occasion and right salpingo-oophorectomy with left salpingectomy on the second. While the precise incidence cannot be determined, rupture from a hemorrhagic corpus luteum appears to be a rare but potentially catastrophic complication of anticoagulant therapy. Hence possible ovarian hemorrhage should be considered in women of reproductive age receiving heparin or sodium warfarin therapy.     

Exposure of the pregnant rat to warfarin and vitamin K1: an animal model of intraventricular hemorrhage in the fetus

Pregnant Sprague-Dawley rats were given daily oral doses of sodium warfarin (100 mg/kg) and concurrent intramuscular injections of vitamin K1 (10 mg/kg). This dosing regimen did not have any apparent deleterious effect on the dams and did not affect the fetuses when administered from day 1 to day 12 of pregnancy. However, similar treatment from day 9 to 20 caused hemorrhage in the fetuses examined on day 21 of gestation. There were no hemorrhages in the control fetuses from dams receiving vitamin K1 only. The lowest effective dose of warfarin, in conjunction with daily doses of vitamin K1, was 3 mg/kg. This dose caused hemorrhage in 28% of fetuses; the incidence of affected fetuses was not further increased by doses of warfarin up to 100 mg/kg. Hemorrhages affected the fetal brain, face, eyes, and ear and occasionally the limbs. Brain hemorrhages were frequently intraventricular and caused various degrees of hydrocephaly. Bony defects were not a feature of prenatal exposure to warfarin. These results show that prenatal exposure of the rat to warfarin and vitamin K duplicates the hemorrhagic abnormalities and pathology associated with prenatal exposure to warfarin in the human. It did not induce bony or facial defects probably because the vitamin K-dependent components of bone development occur postnatally in the rat. This model should allow detailed determination of the role of vitamin K-dependent proteins in development.     

Smad7 Misexpression during Embryonic Angiogenesis Causes Vascular Dilation and Malformations Independently of Vascular Smooth Muscle Cell Function

Numerous in vitro and in vivo studies implicate transforming growth factor-beta (TGFbeta) superfamily signaling in vascular development and maintenance. Mice and humans with mutations in TGFbeta superfamily signaling pathway genes exhibit a range of vascular defects that include dilated, fragile and hemorrhagic vessels, defective angiogenic remodeling, severe vascular malformations including arterio-venous malformations, and disrupted vascular smooth muscle cell recruitment and maintenance. Despite a wealth of data, the functions of TGFbeta superfamily signals during angiogenesis are poorly defined, since early embryonic lethality and difficulty distinguishing between primary and secondary defects frequently confound phenotypic interpretation. To perturb TGFbeta superfamily signaling during angiogenesis, we have misexpressed Smad7, an intracellular antagonist of TGFbeta superfamily signaling, in the developing chick limb and head. We find that the great vessels are strikingly dilated and frequently develop intra and intervascular shunts. Neither noggin nor dominant negative BMP receptor misexpression causes similar vascular phenotypes. However, simultaneous misexpression of constitutively active BMP receptors with Smad7 suppresses the Smad7-induced phenotype, suggesting that a BMP-like intracellular pathway is the target of Smad7 action. Despite the gross morphological defects, further analyses find no evidence of hemorrhage and vessel structure is normal. Furthermore, enlarged vessels and vascular malformations form in either the presence or absence of vascular smooth muscle, and vascular smooth muscle cell recruitment is unperturbed. Our data define the TGFbeta superfamily pathway as an integral regulator of vessel caliber that is also essential for appropriate vessel connectivity. They demonstrate that dilation need not result in vessel rupture or hemorrhage, and dissociate vessel maintenance from the presence of a vascular smooth muscle cell coat. Furthermore they uncouple vascular smooth muscle cell recruitment and differentiation from TGFbeta superfamily signaling.     

CEREBRAL COMPLICATIONS INCURRED DURING PREGNANCY AND THE PUERPERIUM

In a statistical study of maternal mortality cases in Franklin County, Ohio, with a total of 170 deaths in a ten-year period (1948-1957), there were 36 fatal cases with cerebral complications of various types. Intracranial hemorrhage was the cause of death in 17 cases; subarachnoid hemorrhage in eight; intracerebral hemorrhage in eight and subdural hemorrhage in one case. There were nine cases of intracranial tumor with fatality. In a miscellaneous group of ten “cerebral deaths” infectious processes were the cause in eight cases, including tuberculous meningitis, purulent meningitis, brain abscess, acute (cerebromedullary) poliomyelitis, “viral” encephalitis, toxoplasmosis and tetanus.In a smaller clinical (nonfatal) group with cerebral complications occurring during pregnancy and the puerperium, two patients with subarachnoid hemorrhages made spontaneous recovery. A diagnosis of intracerebral hemorrhage was made in three instances, in two of which operation was done and evacuation of blood clots was accomplished. One patient recovered spontaneously from a minimal hemorrhage.Five other persons had cerebral thrombosis, three in the third month of pregnancy and two in the immediate puerperium. All recovered, with some residual deficits.Three patients with intracranial tumor were successfully treated surgically but with disappointing results ultimately (one case each of cerebellar medulloblastoma, cerebral astrocytoma and supratentorial meningioma).Only when the obstetrician, neurologist and the neurosurgeon are fully aware of the signs, symptoms, and many times the rapid course of these cerebral complications of pregnancy, can there be any material lowering of the morbidity and mortality. Emphasis should be placed on the early investigation of all neurological complaints during pregnancy and the puerperium, with immediate institution of an aggressive diagnostic and therapeutic regimen.     

Intracerebral Hemorrhages in Adults with Community Associated Bacterial Meningitis in Adults: Should We Reconsider Anticoagulant Therapy?

Objective

To study the incidence, clinical presentation and outcome of intracranial hemorrhagic complications in adult patients with community associated bacterial meningitis.

Methods

Nationwide prospective cohort study from all hospitals in the Netherlands, from 1 March 2006, through 31 December 2010.

Results

Of the 860 episodes of bacterial meningitis that were included, 24 were diagnosed with intracranial hemorrhagic complications: 8 upon presentation and 16 during clinical course. Clinical presentation between patients with or without intracranial hemorrhage was similar. Causative bacteria were Streptococcus pneumoniae in 16 patients (67%), Staphylococcus aureus in 5 (21%), Pseudomonas aeruginosa and Listeria monocytogenes both in 1 patient (4%). Occurrence of intracranial hemorrhage was associated with death (63% vs. 15%, P<0.001) and unfavorable outcome (94% vs. 34%, P<0.001). The use of anticoagulants on admission was associated with a higher incidence of intracranial hemorrhages (odds ratio 5.84, 95% confidence interval 2.17–15.76).

Conclusion

Intracranial hemorrhage is a rare but devastating complication in patients with community-associated bacterial meningitis. Since anticoagulant therapy use is associated with increased risk for intracranial hemorrhage, physicians may consider reversing or temporarily discontinuing anticoagulation in patients with bacterial meningitis.     

Hemorrhage During Long-Term Anticoagulant Drug Therapy—Part II: Gastrointestinal Hemorrhage

Most of the gastrointestinal hemorrhages occurring during long-term anticoagulant drug therapy of 2,013 patients (reported in the literature) were caused by underlying lesions (44 to 77). Of the 44 lesions, only seven were diagnosed before treatment was started.Most of the episodes of hemorrhage occurred with the prothrombin activity at a so-called “safe” level. Closer investigation before the anticoagulant therapy was begun might have brought the underlying lesion to light.     

Simulation of local cerebral hemorrhage in different brain structures of experimental animals

A standardized experimental model of intracerebral hemorrhagic stroke in small laboratory animals is developed and advanced for chronic neurobiological studies of normal and pathological higher nervous activity as well as disorders developed after acute hemorrhages. A device is advanced which allows a researcher to destroy appropriate brain structures (tissues and local blood vessels) with necessary precision by four-six rotations of curved stereotaxically inserted mandrel-wire knife and, subsequently, to inject autoblood into the area of the lesion. The advanced model is convenient for the reproduction of lesions in different brain regions (for the purpose of experimental knockouts) in neurophysiological, neuropharmacological, and clinical investigations.     

Plasmin cleavage of the amyloid beta-protein: alteration of secondary structure and stimulation of tissue plasminogen activator activity.

Cerebrovascular amyloid beta-protein (A beta) deposition, a key pathological feature of Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis Dutch-type, can lead to intracerebral hemorrhage; however, the mechanism for this remains unclear. Assembled A beta is a potent stimulator of tissue-type plasminogen activator (tPA) in vitro. Herein, we investigated the stimulation of tPA by freshly solubilized A beta 1-40. The rate of tPA stimulation by A beta 1-40 increased dramatically over time, suggesting that A beta may be altered during the course of the reaction. SDS-PAGE analysis showed that A beta 1-40 was cleaved during the course of the reaction. Subsequent studies showed that it was plasmin, the product of tPA activation of plasminogen, that specifically cleaved A beta 1-40 in the amino terminal region between Arg5 and His6. Plasmin effectively cleaved a chromogenic substrate corresponding to this cleavage site in A beta. Circular dichroism spectral analysis showed that A beta 6-40 adopted a strong beta-sheet secondary structure. This truncated A beta 6-40 peptide was a potent stimulator of tPA in vitro. Our results indicate that beta-sheet secondary structure of A beta, which can be promoted by plasmin cleavage, stimulates tPA activity. These findings suggest that pathologic interactions between A beta, tPA, and plasmin in the cerebral vessel wall could result in excessive proteolysis contributing to intracerebral hemorrhages.     

Mechanisms of Vascular Damage by Hemorrhagic Snake Venom Metalloproteinases: Tissue Distribution and In Situ Hydrolysis

Background

Envenoming by viper snakes constitutes an important public health problem in Brazil and other developing countries. Local hemorrhage is an important symptom of these accidents and is correlated with the action of snake venom metalloproteinases (SVMPs). The degradation of vascular basement membrane has been proposed as a key event for the capillary vessel disruption. However, SVMPs that present similar catalytic activity towards extracellular matrix proteins differ in their hemorrhagic activity, suggesting that other mechanisms might be contributing to the accumulation of SVMPs at the snakebite area allowing capillary disruption.

Methodology/Principal Findings

In this work, we compared the tissue distribution and degradation of extracellular matrix proteins induced by jararhagin (highly hemorrhagic SVMP) and BnP1 (weakly hemorrhagic SVMP) using the mouse skin as experimental model. Jararhagin induced strong hemorrhage accompanied by hydrolysis of collagen fibers in the hypodermis and a marked degradation of type IV collagen at the vascular basement membrane. In contrast, BnP1 induced only a mild hemorrhage and did not disrupt collagen fibers or type IV collagen. Injection of Alexa488-labeled jararhagin revealed fluorescent staining around capillary vessels and co-localization with basement membrane type IV collagen. The same distribution pattern was detected with jararhagin-C (disintegrin-like/cysteine-rich domains of jararhagin). In opposition, BnP1 did not accumulate in the tissues.

Conclusions/Significance

These results show a particular tissue distribution of hemorrhagic toxins accumulating at the basement membrane. This probably occurs through binding to collagens, which are drastically hydrolyzed at the sites of hemorrhagic lesions. Toxin accumulation near blood vessels explains enhanced catalysis of basement membrane components, resulting in the strong hemorrhagic activity of SVMPs. This is a novel mechanism that underlies the difference between hemorrhagic and non-hemorrhagic SVMPs, improving the understanding of snakebite pathology.     

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) pathway regulates developmental cerebral-vascular stability via prenylation-dependent signalling pathway

Spontaneous intracranial hemorrhage is a debilitating form of stroke, often leading to death or permanent cognitive impairment. Many of the causative genes and the underlying mechanisms implicated in developmental cerebral-vascular malformations are unknown. Recent in vitro and in vivo studies in mice have shown inhibition of the 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) pathway to be effective in stabilizing cranial vessels. Using a combination of pharmacological and genetic approaches to specifically inhibit the HMGCR pathway in zebrafish (Danio rerio), we demonstrate a requirement for this metabolic pathway in developmental vascular stability. Here we report that inhibition of HMGCR function perturbs cerebral-vascular stability, resulting in progressive dilation of blood vessels, followed by vessel rupture, mimicking cerebral cavernous malformation (CCM)-like lesions in humans and murine models. The hemorrhages in the brain are rescued by prior exogenous supplementation with geranylgeranyl pyrophosphate (GGPP), a 20-carbon metabolite of the HMGCR pathway, required for the membrane localization and activation of Rho GTPases. Consistent with this observation, morpholino-induced depletion of the β-subunit of geranylgeranyltransferase I (GGTase I), an enzyme that facilitates the post-translational transfer of the GGPP moiety to the C-terminus of Rho family of GTPases, mimics the cerebral hemorrhaging induced by the pharmacological and genetic ablation of HMGCR. In embryos with cerebral hemorrhage, the endothelial-specific expression of cdc42, a Rho GTPase involved in the regulation of vascular permeability, was significantly reduced. Taken together, our data reveal a metabolic contribution to the stabilization of nascent cranial vessels, requiring protein geranylgeranylation acting downstream of the HMGCR pathway.     

Triggers and mediators of hemorrhagic transformation in cerebral ischemia

Intracerebral hemorrhagic transformation is a multifactorial phenomenon in which ischemic brain tissue converts into a hemorrhagic lesion with blood-vessel leakage, extravasation, and further brain injury. It has been estimated that up to 30-40% of all ischemic strokes undergo spontaneous hemorrhagic transformation, and this phenomenon may become even more prevalent with the increasing use of thrombolytic stroke therapy. An emerging conceptual model suggests that the loss of microvascular integrity and disruption of neurovascular homeostasis connects the experimental findings of blood-cell extravasation to brain injury after hemorrhage. In this short article, we examine mechanisms related to reperfusion injury and oxidative stress, leukocyte infiltration, vascular activation, and dysregulated extracellular proteolysis as potential triggers of hemorrhagic transformation. Perturbations in cell-cell and cell-matrix signaling within the hypothesized neurovascular unit may ultimately lead to neuroinflammation and apoptotic-like cell death in the parenchyma. Further investigations into the molecular mediators of hemorrhagic transformation may reveal new therapeutic targets for this clinically complex problem.     

磁共振磁敏感加权成像在颅脑疾病中的应用研究

目的探讨磁敏感加权成像在脑部疾病中的临床应用价值。方法对65例临床疑是脑血管病变患者行常规T1WI、T2WI、DWI、SWI序列及增强T1WI、MRA,探讨SWI序列在显示小出血灶、小静脉及含铁血黄素、钙化等顺磁性物质的优越性。结果①海绵状血管瘤,SWI能鉴别出血与血管,发现更多的小出血灶;②动静脉畸形,SWI能够发现更多的细小静脉向大静脉引流;③急性脑梗死,SWI可发现小的出血灶;④脑肿瘤,SWI显示出小的引流静脉;⑤帕金森病,SWI能显示脑内多发异常低信号铁沉积。结论 SWI对低流量血管畸形、小静脉结构、多发细小出血以及铁钙沉积十分敏感,为常规MRI的重要补充,应用于中枢神经系统疾病的诊断和鉴别诊断。     

Pathological developments mediated by cyclophosphamide in rats infected with Trypanosoma lewisi

Trypanosoma lewisi is an obligatory, flagellated parasite of the rat. Despite the fact that naturally the rats overcome the disease, a lethal infection can be induced by the administration of an immunosuppressive agent, i.e. cyclophosphamide (Cy). In the Cy treated infected rats (CyI) the severity of the trypanosome infection was demonstrated in the internal organs, in the following order: lungs > liver > heart > spleen > kidney. The parasites were not detected in the brain. The accumulation of the parasites in the lungs led to the development of hemorrhagic inflammatory foci. The rupture of blood vessels was accompanied by lymphocyte infiltrations into the damaged tissues and multiple foci of edema around the blood vessels. In most cases the lungs were dark brown in color due to intra-alveolar hemorrhages. The spleen of the CyI rats showed general deformation of the tissue's architecture, migration of macrophages and cell depletion due to the Cy action. The liver showed inflammatory hemorrhagic foci associated with massive destruction of the parenchyma. In spite of the heavy parasitemia (> 50%) developed in the CyI rats the brain remained free of parasites, which might explain the non-virulent character of this parasite compared to the African trypanosomes.     

De novo expression of the hemoglobin scavenger receptor CD163 by activated microglia is not associated with hemorrhages in human brain lesions

The main function of CD163 (hemoglobin scavenger receptor) is to bind the hemoglobin-haptoglobin complex, thereby mediating extravasal hemolysis. However, CD163 also has an antiinflammatory function. After CD163-mediated endocytosis, hemoglobin is catabolized further by hemeoxygenase 1 (HO-1). Previously, we found expression of HO-1 to be restricted to microglia/macrophages at sites of hemorrhages in human traumatic and ischemic brain lesions. We now investigated if CD163 expression is also correlated with hemorrhages in brain lesions. Methods. Autopsy brain tissue from 44 cases with hemorrhagic brain lesions (32 traumatic brain injuries/TBI, 12 intracerebral bleedings/ICB), 56 non-hemorrhagic brain lesions (30 ischemias, 26 hypoxias) and 6 control brains were investigated. The post injury survival times ranged from a few minutes to 60 months. Results. In controls, single perivascular monocytes expressed CD163, but only single CD163+ microglia were found in 3/6 cases. CD163+ cells in the parenchyma (activated microglia/macrophages) increased significantly within 24 hours after trauma and ischemia and within 1-7 days following ICB or hypoxia. Overall, significantly lower and higher levels of parenchymal CD163+ cells occurred in hypoxia and ischemia, respectively. Perivascular CD163+ cells also increased significantly in all pathological conditions. In areas remote from circumscribed brain lesions (TBI, ICB, ischemia), significant changes were only found in ICB and ischemia. Conclusions. De novo expression of CD163 by activated microglia/macrophages and CD163+ infiltrating monocytes are neither restricted to nor predominant in hemorrhagic brain lesions. Thus, the antiinflammatory function of CD163 probably predominates, both in hemorrhagic and non-hemorrhagic brain lesions and points to possible immunomodulatory treatment strategies targeting CD163.     

Hemodynamics of ulcerated plaques: before and after

The hemodynamics and fluid mechanical forces in blood vessels have long been implicated in the deposition and growth of atherosclerotic plaque. Detailed information about the hemodynamics in vessels affected by significant plaque deposits can also provide insight into the mechanisms and likelihood of plaque weakening and rupture. In the current study, the governing equations are solved in their finite volume formulation in several patient-specific stenotic geometries. Of specific interest are the flow patterns and forces near ulcerations in the plaque. The flow patterns and forces in vessels with ulcerated plaques are compared with those in stenotic vessels without evidence of ulceration and to the hemodynamics in the same vessels as they likely appeared prior to ulceration. Hemodynamics "before" and "after" hemorrhage may suggest fluid mechanical and morphological factors of diagnostic and predictive value. Recirculation zones, vortex shedding, and secondary flows are captured by both laminar and turbulent solutions. The forces on vessel walls are shown to correlate with unstable plaque deposits. Performing before and after studies of vessels in long-term radiology studies may illuminate mechanisms of hemorrhage and other vessel remodeling.     

Hemorrhage During Long-Term Anticoagulant Drug Therapy—Part IV: Selection and Management of Patients

Most serious hemorrhages that occur during long-term anticoagulant drug therapy are due either to poor patient selection or to poor management of the patient, or both.In each patient being considered for treatment, the risk of bleeding must be evaluated and classified as high, moderate or low.The clinician must especially assess the risk of intracranial hemorrhage in hypertensive patients, and must screen all patients for potential sources of gastrointestinal bleeding. There is ample time for such investigations, since initiating long-term anticoagulant therapy is not an emergency procedure.The desired level of prothrombin activity must be adjusted to the risks determined for each individual patient. There is no single “therapeutic range” applicable to all patients with their varying hemorrhagenic risks.Proper management includes sufficient laboratory testing to maintain the desired prothrombin level, and continued vigilance to detect signs of early bleeding.Preventable hemorrhage cannot be cited as evidence against the value of anticoagulant drug therapy.