23Na chemical shift imaging in the living rat brain using a chemical shift agent, Tm[DOTP]5.

OBJECTIVE: It is well known that the use of shift reagents (SRs) in nuclear magnetic resonance (NMR) studies is substantially limited by an intact blood-brain barrier (BBB). The current study aims to develop a method enabling chemical shift imaging in the living rat brain under physiological conditions using an SR, Tm[DOTP]5-. MATERIALS AND METHODS: Hyperosmotic mannitol bolus injection followed by 60 min infusion of a Tm[DOTP]5- containing solution was administered via a catheter inserted into an internal carotid artery. We monitored the homeostasis of physiological parameters, and we measured the thulium content in brain tissue post mortem using total reflection fluorescence spectroscopy (T-XRF). The alterations of the 23Na resonance spectrum were followed in a 9.4T small animal scanner. RESULTS: Based on the T-XRF measurements, the thulium concentration was estimated at 2.3 ± 1.8 mM in the brain interstitial space. Spectroscopic imaging showed a split of the 23Na resonance peak which became visible 20 min after starting the infusion. Chemical shift imaging revealed a significant decrease of the initial intensity level to 0.915 ± 0.058 at the end of infusion. CONCLUSION: Our novel protocol showed bulk accumulation of Tm[DOTP]5- thus enabling separation of the extra-/intracellular 23Na signal components in the living rat brain while maintaining physiological homeostasis.

Assessing abrasion of orthodontic surface sealants using a modified ophthalmic optical coherence tomography device.

OBJECTIVE: Optical coherence tomography (OCT) is a clinical standard in ophthalmology. Currently, its application in dentistry is gaining increasing interest. In this study, we tested the possibility to use a modified commercially available spectral domain OCT (SD-OCT) to assess the layer thickness of orthodontic surface sealants. MATERIALS AND METHODS: Reference samples of surface sealants for calibration and repeatability testing were measured using a micrometer screw. SD-OCT measurements were compared with micro-CT and light microscopic analyses. After validating the calibration of the SD-OCT, surface sealant layer thickness after aging (thermo cycling) and simulation of professional tooth cleaning (PTC) was assessed using the SD-OCT on 45 extracted teeth assigned to three test groups (n = 15 each): Light Bond™ Sealant, Pro Seal®, and Opal® Seal. RESULTS: SD-OCT showed excellent repeatability and accuracy for measurements of surface sealant layer thickness. Compared with micro-CT, SD-OCT showed better accordance with the reference measurements. The analysis of surface sealants after thermo cycling and PTC revealed poor resistance of Light Bond after only aging and demonstrated substantial wear of all sealants after aging and PTC. CONCLUSION: Imaging using commercially available ophthalmic SD-OCT might represent a suitable non-invasive methodology for longitudinal assessments of surface sealant layer thickness in vitro and in vivo. CLINICAL RELEVANCE: SD-OCT might be a suitable non-invasive method for longitudinal assessments of surface sealant durability in clinical trials.

Responses of retinal and brain microvasculature to streptozotocin induced diabetes revealed by global expression profiling.

This study aims to determine the effects of diabetes in the retinal and brain microvasculature through gene expression profiling. Twelve male Wistar rats were randomly divided into two groups: streptozotocin-induced diabetic rats and time-matched nondiabetic rats. The retinal microvessels (RMVs) and brain microvessels (BMVs) were mechanically isolated from individual rats. Differentially expressed genes (DEGs) in diabetic and nondiabetic microvessels were identified by cDNA microarrays analysis. In RMVs, we identified 43 DEGs, of which 20 were upregulated while 23 were downregulated by diabetes. In BMVs, 35 genes DEGs were identified, of which 22 were upregulated and 13 were downregulated by diabetes. Altered expression of the Nars, Gars, Mars, Iars, Yars, Bcl2, Nqo1, NR4A3, Gpd1, Stc1, Tsc22d3, Tnfrsf21 mRNA as observed in the microarray analyses, was confirmed by quantitative RT-PCR. The aminoacyl-tRNA synthetases (aaRSs) pathway in RMVs was significantly overrepresented as compared to BMVs. Our study demonstrates for the first time that in the brain microvasculature multiple compensatory mechanisms exists, serving to protect brain tissue from diabetic insults, whereas these mechanisms are not activated in the retinal microvasculature. This provides new insights as to why brain microvasculature is less susceptible to diabetes.

The pathophysiology of chronic subdural hematoma revisited: emphasis on aging processes as key factor.

Chronic subdural hematoma (CSH) affects mostly elderly subjects. Previously, pathophysiological concepts suggested that CSH is secondary to degradation of subdural collections of blood and its products exerting merely a mass effect on the underlying brain. During the last decades, however, new insights into the pathogenetic mechanisms urge us to reconsider such a simplistic view. Here, we critically review novel pathophysiological, imaging, interventional, and medical treatment aspects and establish an integrative concept of the pathogenesis of CSH stressing the role of age as key factor. Trauma is considered a trigger event that unleashes a cascade of immunological and angiogenic age-dependent responses. These are associated with hypervascularization of the outer hematoma membrane, rebleeding, and exsudation which are crucial determinants for further development and propagation of CSH. Neurosurgical evacuation of the hematoma has long been thought the only viable treatment option, and it is still the method of choice in the majority of cases. Only more recently, embolization of the middle meningeal artery has been introduced as an alternative to surgery, and pharmacological treatment options are being investigated. Persons with advanced age trauma and other trigger events encounter a repair system with characteristics of senescence. This repair system implies a dysfunctional secretory phenotype of senescent cells and results in an insufficient repair process including chronic inflammation and fibrosis. Increased knowledge about the pathomechanisms of CSH will inform future studies and open new perspectives for its treatment and possibly also for its prevention.

Differential effects of tumor-platelet interaction in vitro and in vivo in glioblastoma.

An elevated platelet count is considered an independent predictor of short survival in glioblastoma and various other tumor entities. Prothrombotic activity of the tumor microcirculation resulting in platelet activation and release of cytokines from activated platelets has been suggested to play a role. This study was designed to analyze the effects of platelet-released cytokines on glioblastoma and endothelial cell proliferation and migration in vitro, and the influence of platelet count on glioblastoma growth and angiogenesis in vivo. In cultured human glioblastoma, umbilical cord and cerebral microvascular endothelial cells platelet-released cytokines significantly stimulated proliferation and migration as well as sprouting and formation of capillary-like structures. In vivo, glioblastoma cells were implanted in mice followed by platelet depletion starting 1 or 8 days later. Tumor volume, proliferative index, and vessel density analyzed 14 days after engraftment did not differ between animals with a normal and a low platelet count. Likewise, no effect of platelet depletion over 20 days upon the volume of intracerebrally growing tumors was observed in mice. Additionally, proliferative activity and vessel density determined in tumor samples from patients operated upon glioblastoma did not show any correlation with the patients' preoperative platelet count. Thus, we conclude that distinct proliferation- and chemotaxis-stimulating effects of platelet-derived cytokines can be achieved in vitro, while the platelet count does not exert a major influence on tumor growth and tumor angiogenesis in GBM in vivo.

Application of micro-CT in small animal imaging.

Over the past decade, the number of publications using micro-computed tomography (muCT) imaging in preclinical in vivo studies has risen exponentially. Higher spatial and temporal resolution are the key technical advancements that have allowed researchers to capture increasingly detailed anatomical images of small animals and to monitor the progression of disease in small animal models. The purpose of this review is to present the technical aspects of muCT, as well as current research applications. Our objectives are threefold: to familiarize the reader with the basics of muCT techniques; to present the type of experimental designs currently used; and to highlight limitations, future directions, in muCT-scanner research applications, and experimental methods. As a first step we present different muCT setups and components, as well as image contrast generation principles. We then present experimental approaches in order of the evaluated organ system. Finally, we provide a short summary of some of the technical limitations of muCT imaging and discuss potential future developments in muCT-scanner techniques and experimental setups.

Vascular imaging in small rodents using micro-CT.

In vivo animal models of neoplasm, stroke, subarachnoid hemorrhage, and other diseases involving alterations in vessel anatomy and diameter, require a fast and easy-to-use imaging tool that captures anatomical structure and biologic function data. Micro-computed tomography angiography (muCTA) offers high spatial and temporal resolution and is suitable to perform this task. However, conducting muCTA in small rodents, especially in mice, requires a high degree of accuracy and precision. This article describes a setup for in vivo muCTA in mice using both a bolus technique with a conventional contrast agent, as well as, angiography with a blood-pool contrast agent. Our setup in mice is at isotropic resolutions up to 16 microm with scanning times less than 1 min. The described protocol also addresses some of the technical challenges associated with the imaging of vascular structures in mice models.

In vivo chlorine-35, sodium-23 and proton magnetic resonance imaging of the rat brain.

In this study we demonstrate the feasibility of combined chlorine-35, sodium-23 and proton magnetic resonance imaging (MRI) at 9.4 Tesla, and present the first in vivo chlorine-35 images obtained by means of MRI. With the experimental setup used in this study all measurements could be done in one session without changing the setup or moving the subject. The multinuclear measurement requires a total measurement time of 2 h and provides morphological (protons) and physiological (sodium-23, chlorine-35) information in one scanning session. Chlorine-35, sodium-23 and high resolution proton images were acquired from a phantom, a healthy rat and from a rat displaying a focal cerebral infarction. Compared to the healthy tissue a signal enhancement of a factor of 2.2 +/- 0.2 in the chlorine-35 and a factor of 2.9 +/- 0.6 in the sodium-23 images is observed in the areas of infarction. Exemplary unlocalized measurement of the in vivo longitudinal and transversal relaxation time of chlorine-35 in a healthy rat showed multi-exponential behaviour. A biexponential fit revealed a fast and a slow relaxing component with T(1,a) = (1.7 +/- 0.4) ms, T(1,b) = (25.1 +/- 1.4) ms, amplitudes of A = 0.26 +/- 0.02, (1-A) = 0.74 +/- 0.02 and T(2,a) = (1.3 +/- 0.1) ms, T(2,b) = (11.8 +/- 1.1) ms, A = 0.64 +/- 0.02, (1-A) = 0.36 +/- 0.02. Combined proton, sodium-23 and chlorine-35 MRI may provide a new approach for non-invasive studies of ionic regulatory processes under physiological and pathological conditions in vivo.

CORMs protect endothelial cells during cold preservation, resulting in inhibition of intimal hyperplasia after aorta transplantation in rats.

Allograft vasculopathy is the leading cause for chronic transplant loss. We investigated if the addition of carbon monoxide releasing molecules (CORMs) to the preservation solution would protect the endothelium from cold preservation injury in an aortic transplantation model. In particular, we tested if CORM preserve vascular functioning and limit neo-intima formation following cold preservation (Cp). Abdominal aortas from Lewis or Fisher rats were subjected to Cp in University of Wisconsin (UW) solution to which 50 µm of CORM-3 was added or not. Hereafter, whole mount staining, acetylcholine mediated vasorelaxation (AMV) and aortic transplantation was performed. In vitro CORM-3 protected human umbilical vein endothelial cells from Cp injury and prevented denudation and intercellular gap formation in aortic grafts. Cp resulted in loss of AMV of aorta segments. By contrast, AMV was preserved after the addition of CORM-3 during Cp. Two months after transplantation Cp of aorta grafts resulted in an increased adventitial remodelling and neo-intima formation. This was significantly blunted by CORM-3 in syngeneic recipients. Our study demonstrates that addition of CORM-3 to UW solution prevents endothelial damage, thereby maintaining vascular function directly after cold preservation. Hence, our findings might offer a novel strategy to prevent vascular damage during CP.

Ultrafast high-resolution in vivo volume-CTA of mice cerebral vessels.

BACKGROUND AND PURPOSE: Animal models developed in rats and mice have become indispensable in preclinical cerebrovascular research. Points of interest include the investigation of the vascular bed and the morphology and function of the arterial, capillary, and venous vessels. Because of their extremely small caliber, in vivo examination of these vessels is extremely difficult. In the present study we have developed a method to provide fast 3D in vivo analysis of cerebral murine vessels using volume computed tomography-angiography (vCTA). METHODS: Using an industrial X-ray inspection system equipped with a multifocus cone beam X-ray source and a 12-bit direct digital flatbed detector, high-speed vCTA (180 degrees rotation in 40 s. at 30 fps) was performed in anesthetized mice. During the scan an iodinated contrast agent was infused via a tail vein. Images were reconstructed using a filtered backprojection algorithm. Image analysis was performed by maximum intensity projection (MIP) and 3D volume reconstruction. RESULTS: All mice tolerated i.v. injection of the iodinated contrast agent well. Smallest achievable voxel size of raw data while scanning the whole neurocranium was 16 mum. Anatomy of cerebral vessels was assessable in all animals, and anatomic differences between mouse strains could easily be detected. Mean vessel diameter was measured in C57BL/6 and BALBc mice. Changes of vessel caliber were assessable by repeated vCTA. CONCLUSIONS: Ultra fast in vivo vCTA of murine cerebral vasculature is feasible at resolutions down to 16 mum. The technique allows the assessment of vessel caliber changes in living mice, thus providing an interesting tool to monitor different features such as vasospasm or vessel patency.

Acid sensing ion channels in rat cerebral arteries: Probing the expression pattern and vasomotor activity.

AIMS: The recent identification of acid sensing ion channels (ASICs) in vascular beds suggests their possible involvement in modulating vasomotor tone. Therefore, we investigated the gene expression profiles of ASIC subtypes in the middle cerebral artery (MCA) of Wistar rats and the functional implication of ASICs in acidosis-induced relaxation as well as maintenance of resting tension. MAIN METHODS: Real time PCR was employed to study the pattern of ASIC mRNA expression in the MCA wall in comparison with (i) matching brain tissue samples and (ii) arteries cultured for 24 h and 48 h. The functional implication regarding vasomotor response to acidosis and maintenance of resting tension was assessed using in vitro myography. KEY FINDINGS: A robust mRNA expression of ASIC-1, -2 and -4 was found in brain tissue samples and to a lower extent in freshly isolated MCA. In the MCA wall, short term culture induced a down-regulation of ASIC-1 and -2 expression without any remarkable change in ASIC-4 expression. Acidosis induced a pH-related relaxation of freshly isolated MCA ring segments, being more pronounced after short term culture. Incubation with the ASIC blocker amiloride moderately enhanced acidosis-induced relaxation, in cultured MCAs somewhat stronger than in freshly isolated vessels. In addition, amiloride resulted in a decrease of resting tension, albeit only in freshly isolated MCA. SIGNIFICANCE: Our results comprehensively describe ASIC subtype composition in the rat MCA in physiological and pathological conditions and strongly suggest the involvement of ASICs in the modulation of vasomotor responses under conditions of normal or decreased pH values.

Hypothermia reduces early hypoperfusion and metabolic alterations during the acute phase of massive subarachnoid hemorrhage: a laser-Doppler-flowmetry and microdialysis study in rats.

Morbidity and mortality of subarachnoid hemorrhage (SAH) are correlated with the severity of the patient's acute neurological deficit. This initial presentation has been attributed to cerebral hypoperfusion in the acute phase, and we investigated the impact of moderate hypothermia on the early changes in perfusion and metabolism following massive experimental SAH. SAH was induced in 61 anesthetized rats by rapid injection of 0.5 mL of arterial blood into the cisterna magna. In normothermia (NT), animals were kept at 37 degrees C, while in the primary hypothermia (pHT) group, temperature was lowered to 32 degrees C prior to SAH, and in the secondary hypothermia (sHT) group, cooling was started immediately after SAH. From 30 min prior to 180 min after SAH, Laser-Doppler-flowmetry (LDF) probes allowed online recording of cerebral blood flow (CBF) while parenchymal dialysate was collected by microdialysis probes within the frontoparietal cortex. In NT, the acute phase was characterized by impaired autoregulation and prolonged hypoperfusion. In pHT and sHT, autoregulation was preserved and acute hypoperfusion rapidly improved. SAH also caused a highly significant reduction in glucose in NT only. pHT significantly reduced accumulation of lactate, glutamate, and aspartate. Comparable trends were present for histidine, GABA, and taurine, while glutamine consumption was ameliorated. Early perfusion deficits caused by acute hypoperfusion and disruption of cerebral autoregulation can be ameliorated by hypothermia. Also, the acute phase of experimental SAH is characterized by glucose depletion, lactate accumulation, and release of excitatory amino acids, which can be influenced favorably by hypothermia.

Hypothermia reduces cytotoxic edema and metabolic alterations during the acute phase of massive SAH: a diffusion-weighted imaging and spectroscopy study in rats.

Acute changes in cerebral perfusion and metabolism after subarachnoid hemorrhage (SAH) have been shown to contribute significantly to acute brain injury. The purpose of this study was to examine the effects of moderate hypothermia on the acute changes after massive experimental SAH as evaluated by diffusion-weighted imaging (DWI) and magnetic resonance spectroscopy (MRS). SAH in rats was induced by injection of 0.5 mL of arterial blood. Normothermic animals (NT, n = 10) were kept at 37.0 +/- 0.2 degrees C, while temperature was lowered to 32.0 +/- 0.2 degrees C in the primary hypothermia group (pHT, n = 10) prior to SAH and in the secondary hypothermia group (sHT, n = 10) immediately after SAH. DWI and MRS were performed from 30 min prior up to 3 h after injury. The apparent diffusion coefficient (ADC) was measured in cortical and hippocampal regions of interest (ROIs). MRS included lactate, N-acetyl aspartate (NAA), and creatine in a central voxel. DWI showed a generalized, significant decline in ADC after SAH in NT. Significant change in ADC in pHT was absent, and accelerated recovery for animals in sHT was noted. MRS analysis revealed significant lactate accumulation to 204 +/- 40% from baseline only in NT, while sHT was characterized by a transient, less pronounced increase of lactate (159 +/- 11%) and lactate in pHT did not change significantly (117 +/- 11%). NAA did not change significantly when compared to baseline or between groups for NT, pHT, or sHT. Creatine rose significantly to 166 +/- 27% in NT after the insult, indicating increased metabolic stress which was absent in pHT (106 +/- 8%) and sHT (124 +/- 18%). Hypothermia can ameliorate early development of cytotoxic edema, lactate accumulation, and a general metabolic stress response after SAH, even when started after the insult. Our study indicates that a potentially beneficial influence on metabolism and cerebral perfusion in this crucial phase is practicable and might hold the key to further improve outcome in SAH.

Clazosentan, an endothelin receptor antagonist, prevents early hypoperfusion during the acute phase of massive experimental subarachnoid hemorrhage: a laser Doppler flowmetry study in rats.

OBJECT: Acute cerebral hypoperfusion and early disturbances in cerebral autoregulation after subarachnoid hemorrhage (SAH) have been demonstrated repeatedly and have been shown to contribute significantly to acute and secondary brain injury. Acute vasoconstriction has been identified as a major contributing factor. Although increasing evidence implicates endothelin (ET)-1 in the development of cerebral vasospasm, its role in the acute phase after SAH has not yet been investigated. The purpose of this study was to further determine the role of ET in the first minutes to hours after massive experimental SAH induced by prophylactic treatment with the ET receptor antagonist clazosentan. METHODS: Subarachnoid hemorrhage was induced in 22 anesthetized rats by injection of 0.5-ml arterial, nonheparinized blood into the cisterna magna over the course of 60 seconds. In addition to monitoring intracranial pressure (ICP) and mean arterial blood pressure, laser Doppler flowmetry (LDF) probes were placed stereotactically over the cranial windows to allow online recording of cerebral blood flow (CBF) starting 30 minutes prior to SAH and continuing for 3 hours after SAH. The control group (Group A, 11 rats) received vehicle saline solution via a femoral catheter before SAH, and a second group (Group B, 11 rats) was treated prophylactically with clazosentan, an ET(A) receptor antagonist. Treatment was started 30 minutes prior to bolus injection (1 mg/kg body weight), immediately followed by a continuous infusion of 1 mg/kg body weight/hr until the end of the experiment. RESULTS: Induction of SAH in the rats caused an immediate increase in ICP, which led to an acute decrease in cerebral perfusion pressure (CPP). Perfusion, as measured with LDF, was found to have decreased relative to baseline by 30 +/-20% in the control group and 20 +/-9% in the clazosentan-treated group. Intracranial pressure and CPP recovered comparably in both groups thereafter within minutes. Control animals demonstrated prolonged hypoperfusion with a loss of autoregulation independent of CPP changes, finally approaching 80% of baseline values toward the end of the experiment. The authors observed that clazosentan did not influence peracute CPP-dependent hypoperfusion, but prevented continuous CBF reduction. Laser Doppler flowmetry perfusion readings remained depressed in control animals at 73 +/-19% of baseline in comparison with 106 +/-25% of baseline in clazosentan-treated animals (p = 0.001). CONCLUSIONS: The first hours after a massive experimental SAH can be characterized by a CPP-independent compromise in cerebral perfusion. Prophylactic treatment with the ET receptor antagonist clazosentan prevented hypoperfusion. It is known that in the first days after SAH, a reduction in CBF correlates clinically to high-grade SAH. Although research currently focuses on delayed vasospasm, administration of vasoactive drugs in the acute phase of SAH may reverse perfusion deficits and improve patient recovery.

Enhancement of bradykinin-induced relaxation by focal brain ischemia in the rat middle cerebral artery: Receptor expression upregulation and activation of multiple pathways.

Focal brain ischemia markedly affects cerebrovascular reactivity. So far, these changes have mainly been related to alterations in the level of smooth muscle cell function while alterations of the endothelial lining have not yet been studied in detail. We have, therefore, investigated the effects of ischemia/reperfusion injury on bradykinin (BK)-induced relaxation since BK is an important mediator of tissue inflammation and affects vascular function in an endothelium-dependent manner. Focal brain ischemia was induced in rats by endovascular filament occlusion (2h) of the middle cerebral artery (MCA). After 22h reperfusion, both MCAs were harvested and the response to BK studied in organ bath experiments. Expression of the BK receptor subtypes 1 and 2 (B1, B2) was determined by real-time semi-quantitative RT-qPCR methodology, and whole mount immunofluorescence staining was performed to show the B2 receptor protein expression. In control animals, BK did not induce significant vasomotor effects despite a functionally intact endothelium and robust expression of B2 mRNA. After ischemia/reperfusion injury, BK induced a concentration-related sustained relaxation in all arteries studied, more pronounced in the ipsilateral than in the contralateral MCA. The B2 mRNA was significantly upregulated and the B1 mRNA displayed de novo expression, again more pronounced ipsi- than contralaterally. Endothelial cells displaying B2 receptor immunofluorescence were observed scattered or clustered in previously occluded MCAs. Relaxation to BK was mediated by B2 receptor activation, abolished after endothelium denudation, and largely diminished by blocking nitric oxide (NO) release or soluble guanylyl cyclase activity. Relaxation to BK was partially inhibited by charybdotoxin (ChTx), but not apamin or iberiotoxin suggesting activation of an endothelium-dependent hyperpolarization pathway. When the NO-cGMP pathway was blocked, BK induced a transient relaxation which was suppressed by ChTx. After ischemia/reperfusion injury BK elicits endothelium-dependent relaxation which was not detectable in control MCAs. This gain of function is mediated by B2 receptor activation and involves the release of NO and activation of an endothelium-dependent hyperpolarization. It goes along with increased B2 mRNA and protein expression, leaving the functional role of the de novo B1 receptor expression still open.

A novel method to isolate retinal and brain microvessels from individual rats: Microscopic and molecular biological characterization and application in hyperglycemic animals.

Alterations in the retinal microvessel (RMV) compartment occurring in systemic disease states such as diabetes may eventually contribute to blindness. To specifically address the pathophysiological role of the microvasculature we developed a new method for RMV bulk isolation from individual rats. The extraction procedure performed in the cold throughout takes less than one hour. Slight modifications enable isolation of brain microvessels (BMVs) for comparison. Microscopically, RMVs and BMVs consisted mainly of capillaries of good structural integrity. The endothelial cell/pericyte ratio was approximately 1.8 in RMVs and 2.7 in BMVs, well in agreement with data from intact vascular beds. Total RNA extracted from individual rats amounted to approximately 7 ng in RMVs, 50 ng in BMVs, and 155 ng in pial arteries (which were also isolated) with highly preserved integrity throughout. Measurements using microfluidic card methodology revealed segregation of RMVs, BMVs, and pial arteries in distinct clusters based on principal component analysis. In all three vascular compartments endothelial cell-specific markers were significantly enriched. Similarly, pericyte-specific markers displayed accumulation in RMVs, BMVs, and pial arteries, the latter probably reflecting the common ontogenetic origin of pericytes and smooth muscle cells. Isolation of RMVs, BMVs, and pial arteries from rats suffering from 8-weeks hyperglycemia yielded expression patterns of endothelial cell- and pericyte-specific marker genes largely comparable to those obtained in control rats. Our newly developed protocols allow for selective studies of RMVs from individual rats to characterize reactive pathways, in comparison with the ontogenetically closely related BMVs. Moreover, our protocols with inclusion of pial arteries enable comparative studies of the macro- and microvasculature from the same organ.

Granulocyte-macrophage colony-stimulating factor-induced vessel growth restores cerebral blood supply after bilateral carotid artery occlusion.

BACKGROUND AND PURPOSE: Hemodynamic compromise due to occlusive cerebrovascular disease is associated with an increased stroke risk. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been suggested to stimulate collateral blood vessel growth in various models of hemodynamic compromise. The purpose of this study was to investigate the effects of GM-CSF on cerebral hemodynamics and vessel growth in a rat model of chronically impaired cerebral blood flow (CBF). METHODS: Male Sprague-Dawley rats underwent sequential bilateral carotid artery occlusion (BCO) and were treated with GM-CSF or saline for 6 weeks. Sham-occluded animals served as a control group. Baseline CBF was measured by iodo[(14)C]antipyrine autoradiography, and cerebrovascular reserve capacity was assessed by laser-Doppler flowmetry after application of 20 mg/kg body weight acetazolamide. The capillary density and arterioles immunopositive for alpha-smooth muscle actin were counted on brain sections. The cerebral angioarchitecture was visualized with a latex perfusion technique. RESULTS: Baseline CBF as measured by iodo[(14)C]antipyrine autoradiography was not affected by BCO. The cerebrovascular reserve capacity, however, was significantly impaired 1 week after BCO. CBF and cerebrovascular reserve capacity recovered completely in GM-CSF-treated animals but not in solvent-treated animals. Histologic analysis of the hippocampus revealed integrity of the hypoxia-vulnerable neurons in all animals. The capillary density showed a very mild increase in GM-CSF-treated animals. However, the number of intraparenchymal and leptomeningeal arterioles was significantly higher in GM-CSF-treated animals than in both other groups. CONCLUSIONS: Long-term GM-CSF treatment in a BCO model in rats leads to restoration of impaired cerebral hemodynamics and accompanies structural changes in the resistance-vessel network.

A new method for superselective middle cerebral artery infusion in the rat.

OBJECT: Selective intraarterial drug delivery is used to achieve enhanced local uptake with reduced systemic side effects. In the present paper the authors describe and characterize a new microcatheter-based model of superselective perfusion of the middle cerebral artery (MCA) in rats combined with blockade of blood flow through the MCA. METHODS: Selectivity of administration was shown by infusion of Evans blue which diffusely stained the MCA territory, indicating an increased permeability of the blood-brain barrier during the blockade of blood flow to the MCA. Perfusion of autologous blood through the microcatheter resulted in a flow rate-related increase in the cerebral blood flow measured by laser Doppler flowmetry. Similarly, infusion of an artificial O2 carrier, Oxycyte, was accompanied by an increase in tissue oxygenation as measured using a Licox sensor. Blockade of blood flow to the MCA with the new microcatheter for an extended period of time resulted in the development of ischemia, which was comparable to that induced by intravascular occlusion using a silicone-coated thread. In a 24-hour MCA occlusion model, selective administration of a low dose of MK-801 (0.3 mg/kg body weight) resulted in a significantly smaller infarct volume than systemic application (339 +/- 53 mm(3) compared with 508 +/- 26 mm(3), p < 0.001). CONCLUSIONS: This new model of superselective MCA infusion is a valuable tool for investigating the effect of selective delivery and enhanced drug uptake into cerebral ischemic tissue. Without constant blockade of blood flow through the MCA it may also be useful for enhanced drug uptake, gene transfer, or application of stem cells in other neuropathological conditions.

Comparison of different intravascular thread occlusion models for experimental stroke in rats.

For the induction of ischemic strokes of varying sizes in rats, different types of threads were used to occlude the middle cerebral artery (MCA) in combination with or without the posterior communicating artery (PCOM) and the common carotid artery (CCA). During vessel occlusion brain tissue partial oxygen pressure (ptiO2) and regional cerebral blood flow (rCBF) were monitored using a combined ptiO2/laser Doppler flow probe. Following neurological assessment animals were sacrificed at 3, 8 and 24 h and the necrotic volume was measured on serial coronary slices. In another experimental group, rCBF was measured 1h post-insult with the iodo[14C]antipyrine method. Animals with selective MCA occlusion showed less reduction of ptiO2 and rCBF and smaller infarcts when compared with animals with combined occlusion of the MCA, CCA and PCOM. Both groups, selective MCA occlusion and combined occlusion of the MCA, CCA and PCOM, demonstrated a high reproducibility and low variability of stroke size. Relative growth of stroke size within 24 h was higher in animals with selective MCA occlusion. Therefore, the selective MCAO model may be advantageous for studies of neuroprotective strategies.

Long-term survival in permanent middle cerebral artery occlusion: a model of malignant stroke in rats.

Occlusion of the middle cerebral artery (MCA) by an intraluminal filament is widely used to study focal brain ischemia in male Sprague-Dawley rats. However, permanent occlusion goes along with a high fatality. To overcome this drawback we designed a new filament carrying a bowling pin-shaped tip (BP-tip) and compared this with three conventionally tipped filaments. Follow-up periods were 24 h (all groups) and 72 and 120 h in BP-tip group. Ischemic damage and swelling were quantified using silver nitrate staining. Collateral flow via the posterior cerebral artery (PCA) was assessed using selective dye perfusion of the internal carotid artery. Despite a comparable decrease of brain perfusion in all groups, ischemic damage was significantly smaller in BP-tips (p < 0.05). Moreover, BP-tip significantly reduced mortality from 60% to 12.5% and widely spared the occipital region and hypothalamus from ischemic damage. Conventional but not BP-tip filaments induced vascular distortion, measured as gross displacement of the MCA origin, which correlated with occipital infarction size. Accordingly, BP-tip occluded rats showed a significantly better collateral filling of the PCA territory. Ischemic volume significantly increased in BP-tip occlusion at 72 h follow-up. BP-tip filaments offer superior survival in permanent MCA occlusion, while mimicking the course of a malignant stroke in patients.