A surgical model of permanent and transient middle cerebral artery stroke in the sheep

Background

Animal models are essential to study the pathophysiological changes associated with focal occlusive stroke and to investigate novel therapies. Currently used rodent models have yielded little clinical success, however large animal models may provide a more suitable alternative to improve clinical translation. We sought to develop a model of acute proximal middle cerebral artery (MCA) ischemic stroke in sheep, including both permanent occlusion and transient occlusion with reperfusion.

Materials and Methods

18 adult male and female Merino sheep were randomly allocated to one of three groups (n = 6/gp): 1) sham surgery; 2) permanent proximal MCA occlusion (MCAO); or 3) temporary MCAO with aneurysm clip. All animals had invasive arterial blood pressure, intracranial pressure and brain tissue oxygen monitoring. At 4 h following vessel occlusion or sham surgery animals were killed by perfusion fixation. Brains were processed for histopathological examination and infarct area determination. 6 further animals were randomized to either permanent (n = 3) or temporary MCAO (n = 3) and then had magnetic resonance imaging (MRI) at 4 h after MCAO.

Results

Evidence of ischemic injury in an MCA distribution was seen in all stroke animals. The ischemic lesion area was significantly larger after permanent (28.8%) compared with temporary MCAO (14.6%). Sham animals demonstrated no evidence of ischemic injury. There was a significant reduction in brain tissue oxygen partial pressure after permanent vessel occlusion between 30 and 210 mins after MCAO. MRI at 4 h demonstrated complete proximal MCA occlusion in the permanent MCAO animals with a diffusion deficit involving the whole right MCA territory, whereas temporary MCAO animals demonstrated MRA evidence of flow within the right MCA and smaller predominantly cortical diffusion deficits.

Conclusions

Proximal MCAO can be achieved in an ovine model of stroke via a surgical approach. Permanent occlusion creates larger infarct volumes, however aneurysm clip application allows for reperfusion.     

Mild Sensory Stimulation Completely Protects the Adult Rodent Cortex from Ischemic Stroke

Despite progress in reducing ischemic stroke damage, complete protection remains elusive. Here we demonstrate that, after permanent occlusion of a major cortical artery (middle cerebral artery; MCA), single whisker stimulation can induce complete protection of the adult rat cortex, but only if administered within a critical time window. Animals that receive early treatment are histologically and behaviorally equivalent to healthy controls and have normal neuronal function. Protection of the cortex clearly requires reperfusion to the ischemic area despite permanent occlusion. Using blood flow imaging and other techniques we found evidence of reversed blood flow into MCA branches from an alternate arterial source via collateral vessels (inter-arterial connections), a potential mechanism for reperfusion. These findings suggest that the cortex is capable of extensive blood flow reorganization and more importantly that mild sensory stimulation can provide complete protection from impending stroke given early intervention. Such non-invasive, non-pharmacological intervention has clear translational potential.     

Development of a middle cerebral artery occlusion model in the nonhuman primate and a safety study of i.v. infusion of human mesenchymal stem cells

Background

Most experimental stroke research is carried out in rodents, but given differences between rodents and human, nonhuman primate (NHP) models may provide a valuable tool to study therapeutic interventions. The authors developed a surgical method for transient occlusion of the M1 branch of middle cerebral artery (MCA) in the African green monkey to evaluate safety aspects of intravenous infusion of mesenchymal stem cells (hMSCs) derived from human bone marrow.

Methods

The left Sylvian fissure was exposed by a small fronto-temporal craniotomy. The M1 branch of the MCA was exposed by microsurgical dissection and clipped for 2 to 4 hours. Neurological examinations and magnetic resonance imaging (MRI) were carried out at regular post-operative course. hMSCs were infused 1 hour after reperfusion (clip release) in the 3-hour occlusion model.

Results

During M1 occlusion, two patterns of changes were observed in the lateral hemisphere surface. One pattern (Pattern 1) was darkening of venous blood, small vessel collapse, and blood pooling with no venous return in cortical veins. Animals with these three features had severe and lasting hemiplegia and MRI demonstrated extensive MCA territory infarction. Animals in the second pattern (Pattern 2) displayed darkening of venous blood, small vessel collapse, and reduced but incompletely occluded venous flow and the functional deficit was much less severe and MRI indicated smaller infarction areas in brain. The severe group (Pattern 1) likely had less extensive collateral circulation than the less severe group (Pattern 2) where venous pooling of blood was not observed. The hMSC infused animals showed a trend for greater functional improvement that was not statistically significant in the acute phase and no additive negative effects.

Conclusions

These results indicate inter-animal variability of collateral circulation after complete M1 occlusion and that hMSC infusion is safe in the developed NHP stroke model.     

超顺磁性造影剂的研究进展

超顺磁性造影剂是一种新型的磁共振造影剂,对肝脾等部位的成像效果显著,已成为国内外研究的热点之一。本文对超顺磁性造影剂的增强原理和制备方法进行了总结及评论。     

MRI detection of hyperoxia-induced lung edema in Zn-deficient rats

In a pioneering application of proton Magnetic Resonance Imaging (MRI), lung edema has been monitored in vivo in Zn-deficient rats exposed to 85% oxygen. Dietary Zn appears to play a role in protecting against hyperoxia-induced lung damage.     

Non-rigid deformation to include subject-specific detail in musculoskeletal models of CP children with proximal femoral deformity and its effect on muscle and contact forces during gait

To account for proximal femoral deformities in children with cerebral palsy (CP), subject-specific musculoskeletal models are needed. Non-rigid deformation (NRD) deforms generic onto personalized bone geometry and thereby transforms the muscle points. The goal of this study was to determine to what extent the models and simulation outcomes in CP patients differ when including subject-specific detail using NRD or Magnetic Resonance Imaging (MRI)-based models. The NRD models slightly overestimated hip contact forces compared to MRI models and differences in muscle point positions and moment arm lengths (MALs) remained, although differences were smaller than for the generic model.     

Effect of endothelin antagonist (TAK-044) on cerebral ischemic volume, oxidative stress markers and neurobehavioral parameters in the middle cerebral artery occlusion model of stroke in rats

Stroke causes brain injury in millions of people world wide each year. Despite the enormity of problem, currently there is no established therapy, which can restore the blood flow at infracted area and also improve the neurological deficit. The present study was carried out to investigate the effect of an endothelin antagonist (TAK-044) in middle cerebral artery (MCA) occlusion model of acute ischemic stroke in rats. Male Wistar rats were pretreated with TAK-044 (5 mg/kg, i.p.) for 7 days and thereafter subjected to focal ischemia by occlusion of MCA using intraluminal thread for two hours. 30 min after reperfusion the animals were subjected to diffusion-weighted imaging (DWI) for assessment of protective effect. Twenty-four hours later the motor performance was tested and subsequently the animals were sacrificed for estimation of markers of oxidative stress; malondialdehyde (MDA), glutathione (GSH) and superoxide dismutase (SOD). Control group received vehicle (saline) and similar experimental protocol was followed. In the TAK-044 pretreated group, percent hemispheric lesion area (% HLA) in DWI was significantly attenuated 17.5 +/- 0.5% as compared to control group 61.2 +/- 5.9%. Significant motor impairment, with significant elevated levels of MDA, decrease in GSH and SOD were observed in the vehicle treated MCA occluded rats. Pretreatment with TAK-044 prevented the motor impairment and significantly reversed the changes in markers of oxidative stress (MDA, GSH and SOD). In addition to well-known vasodilatory effect, TAK-044 has recently been documented to have antioxidant and anti-inflammatory properties. These effects can contribute to the protection afforded by TAK-044 in the present study.     

Speech MRI: Morphology and function

Magnetic Resonance Imaging (MRI) plays an increasing role in the study of speech. This article reviews the MRI literature of anatomical imaging, imaging for acoustic modelling and dynamic imaging. It describes existing imaging techniques attempting to meet the challenges of imaging the upper airway during speech and examines the remaining hurdles and future research directions.     

Melatonin reduces cerebral edema formation caused by transient forebrain ischemia in rats

Reduction of cerebral edema, an early symptom of ischemia, is one of the most important remedies for reducing subsequent chronic neural damage in stroke. Melatonin, a metabolite of tryptophan released from the pineal gland, has been found to be effective against neurotoxicity in vitro. The present study was aimed to demonstrate the effectiveness of melatonin in vivo in reducing ischemia-induced edema using magnetic resonance imaging (MRI). Rats were subjected to middle cerebral artery (MCA) occlusion/reperfusion surgery. Melatonin was administered twice (6.0 mg/kg, p.o.): just prior to 1 h MCA occlusion and 1 day after the surgery. T2-weighted multislice spin-echo images were acquired 1 day after the surgery. Increases in T2-weighted signals in ischemic sites of the brain were clearly observed after MCA occlusion. The signal increase was found mainly in the striatum and in the cerebral cortex in saline-treated control rats. In the melatonin-treated group, the total volume of cerebral edema was reduced by 45.3% compared to control group (P < 0.01). The protective effect of melatonin against cerebral edema was more clearly observed in the cerebral cortex (reduced by 56.1%, P < 0.01), while the reduction of edema volume in the striatum was weak (reduced by 23.0%). The present MRI study clearly demonstrated that melatonin is effective in reducing edema formation in ischemic animals in vivo, especially in the cerebral cortex. Melatonin may be highly useful in preventing cortical dysfunctions such as motor, sensory, memory, and psychological impairments.     

奇异值分解在磁共振波谱成像中的应用

目的：磁共振波谱成像有7类快速方法,它们都来自快速MRI成像方法。本文提出的奇异值分解波谱成像不同这7类快速方法,它是把MRI中任意轨迹图像重建方法用于波谱成像,这将有利于设计出速度更快的波谱成像数据采集脉冲序列。     

The protective effects of T cell deficiency against brain injury are ischemic model-dependent in rats

Previous studies have reported that T cell deficiency reduced infarct sizes after transient middle cerebral artery (MCA) suture occlusion in mice. However, how reperfusion and different models affect the detrimental effects of T cells have not been studied. We investigated the effects of T cell deficiency in nude rats using two stroke models and compared their infarct sizes with those in WT rats. In the distal MCA occlusion (MCAo) model, the distal MCA was permanently occluded and the bilateral common carotid arteries (CCAs) were transiently occluded for 60 min. In the suture MCAo model, the MCA was transiently occluded for 100 min by the insertion of a monofilament suture. Our results showed that T cell deficiency resulted in about a 50% reduction in infarct size in the suture MCAo model, whereas it had no effect in the distal MCAo model, suggesting the protective effects of T cell deficiency are dependent on the ischemic model used. We further found more total T cells, CD4 T cells and CD8 T cells in the ischemic brains of WT rats in the suture MCAo model than in the distal MCAo model. In addition, we detected more CD68-expressing macrophages in the ischemic brains of WT rats than in nude rats in the suture MCAo but not the distal MCAo model. Lymphocyte reconstitution in nude rats resulted in larger infarct sizes in the suture MCAo, but not in the distal MCAo stroke model. The results of regional CBF measurement indicated a total reperfusion in the MCAo model but only a partial reperfusion in the distal MCAo model. In conclusion, the protective effects of T cell deficiency on brain injury are dependent on the ischemic model used; likely associated with different degrees of reperfusion.     

Characterizing Photothrombotic Distal Middle Cerebral Artery Occlusion and YAG Laser-Induced Reperfusion Model in the Izumo Strain of Spontaneously Hypertensive Rats

No study has systematically studied the relevance of original Izumo strain of spontaneously hypertensive rats (SHR/Izm) as a stroke model. Furthermore, both SHR/Izm and stroke-prone SHR/Izm (SHRSP/Izm) are commercially available, and recent progress in genetic studies allowed us to use several congenic strains of rats constructed with SHR/Izm and SHRSP/Izm as the genetic background strains. A total of 166 male SHR/Izm and 17 male SHRSP/Izm were subjected to photothrombotic middle cerebral artery (MCA) occlusion with or without YAG laser-induced reperfusion. The pattern of distal MCA was recorded. Infarct volumes were determined with 2,3,5-triphenyltetrazolium chloride. At 24 or 48 h after MCA occlusion, infarct volumes in the permanent occlusion and 2-h occlusion groups (88 ± 22 [SD] and 87 ± 25 mm³, respectively) were significantly larger than that in the 1-h occlusion group (45 ± 14 mm³), indicating the presence of sizeable zone of penumbra. Infarct size in SHRSP/Izm determined at 24 h after MCA occlusion was fairly large (124.0 ± 34.8 mm³, n = 10). Infarct volume in SHR/Izm with simple distal MCA was 76 ± 19 mm³, which was significantly smaller than 95 ± 22 mm³ in the other SHR/Izm with more branching MCA. These data suggest that this stroke model in SHR/Izm is useful in the preclinical testing of stroke therapies and elucidating the pathophysiology of cerebral ischemia/reperfusion.     

High-Intensity Focused Ultrasound (HIFU) for Dissolution of Clots in a Rabbit Model of Embolic Stroke

It is estimated that only 2-6% of patients receive thrombolytic therapy for acute ischemic stroke suggesting that alternative therapies are necessary. In this study, we investigate the potential for high intensity focused ultrasound (HIFU) to initiate thrombolysis in an embolic model of stroke. Iron-loaded blood clots were injected into the middle cerebral artery (MCA) of New Zealand White rabbits, through the internal carotid artery and blockages were confirmed by angiography. MRI was used to localize the iron-loaded clot and target the HIFU beam for treatment. HIFU pulses (1.5 MHz, 1 ms bursts, 1 Hz pulse repetition frequency, 20 s duration) were applied to initiate thrombolysis. Repeat angiograms and histology were used to assess reperfusion and vessel damage. Using 275 W of acoustic power, there was no evidence of reperfusion in post-treatment angiograms of 3 rabbits tested. In a separate group of animals, 415 W of acoustic power was applied and reperfusion was observed in 2 of the 4 (50%) animals treated. In the last group of animals, acoustic power was further increased to 550 W, which led to the reperfusion in 5 of 7 (～70%) animals tested. Histological analysis confirmed thatthe sonicated vessels remained intact after HIFU treatment. Hemorrhage was detected outside of the sonication site, likely due to the proximity of the target vessel with the base of the rabbit skull. These results demonstrate the feasibility of using HIFU, as a stand-alone method, to cause effective thrombolysis without immediate damage to the targeted vessels. HIFU, combined with imaging modalities used to identify and assess stroke patients, could dramatically reduce the time to achieve flow restoration in patients thereby significantly increasing the number of patients which benefit from thrombolysis treatments.     

A Novel Procedure for Rapid Imaging of Adult Mouse Brains with MicroCT Using Iodine-Based Contrast

High-resolution Magnetic Resonance Imaging (MRI) has been the primary modality for obtaining 3D cross-sectional anatomical information in animals for soft tissue, particularly brain. However, costs associated with MRI can be considerably high for large phenotypic screens for gross differences in the structure of the brain due to pathology and/or experimental manipulations. MicroCT (mCT), especially benchtop mCT, is becoming a common laboratory equipment with throughput rates equal or faster than any form of high-resolution MRI at lower costs. Here we explore adapting previously developed contrast based mCT to image adult mouse brains in-situ. We show that 2% weight per volume (w/v) iodine-potassium iodide solution can be successfully used to image adult mouse brains within 48 hours post-mortem when a structural support matrix is used. We demonstrate that hydrogel can be effectively used as a perfusant which limits the tissue shrinkage due to iodine.     

Protection against focal cerebral ischemia following exposure to a pulsed electromagnetic field

There is evidence that electromagnetic stimulation may accelerate the healing of tissue damage following ischemia. We undertook this study to investigate the effects of low frequency pulsed electromagnetic field (PEMF) exposure on cerebral injury in a rabbit model of transient focal ischemia (2 h occlusion followed by 4 h of reperfusion). PEMF exposure (280 V, 75 Hz, IGEA Stimulator) was initiated 10 min after the onset of ischemia and continued throughout reperfusion (six exposed, six controls). Magnetic resonance imaging (MRI) and histology were used to measure the degree of ischemic injury. Exposure to pulsed electromagnetic field attenuated cortical ischemic edema on MRI at the most anterior coronal level by 65% (P < 0.001). On histologic examination, PEMF exposure reduced ischemic neuronal damage in this same cortical area by 69% (P < 0.01) and by 43% (P < 0.05) in the striatum. Preliminary data suggest that exposure to a PEMF of short duration may have implications for the treatment of acute stroke. © 1994 Wiley-Liss, Inc.     

Derivatives of thiocolchicine and its applications to CEM cells treatment using F Magnetic Resonance ex vivo

It was shown, that cultured ex vivo human T-Lymphoblastoid (CEM) cells respond to synthesized thiocolchicine and fluorine thiocolchicine derivatives. The preparation of derivatives with substitution at C-3 and C-7 is described. All compounds were used at concentration from 1 nM to 1000 nM. Inhibitory effects of these compounds were examined in the three-dimensional (3-D) culture and cells morphology during treatment was monitored using 9.4 T MRI system. We performed studies of these compounds in CEM cells ex vivo using ¹H and ¹⁹F Magnetic Resonance Imaging (MRI), ¹⁹F Magnetic Resonance Spectroscopy (MRS), High Performance Liquid Chromatography coupled with Ultra Violet (HPLC-UV) and Electron Impact Mass Spectrometry (EIMS). The results of the multi-technique approach are consistent with the fact that the new derivatives are more efficient than colchicine and thiocolchicine ex vivo.     

Elevated Intracranial Pressure and Cerebral Edema following Permanent MCA Occlusion in an Ovine Model

Introduction

Malignant middle cerebral artery (MCA) stroke has a disproportionately high mortality due to the rapid development of refractory space-occupying cerebral edema. Animal models are essential in developing successful anti-edema therapies; however to date poor clinical translation has been associated with the predominately used rodent models. As such, large animal gyrencephalic models of stroke are urgently needed. The aim of the study was to characterize the intracranial pressure (ICP) response to MCA occlusion in our recently developed ovine stroke model.

Materials and Methods

30 adult female Merino sheep (n = 8–12/gp) were randomized to sham surgery, temporary or permanent proximal MCA occlusion. ICP and brain tissue oxygen were monitored for 24 hours under general anesthesia. MRI, infarct volume with triphenyltetrazolium chloride (TTC) staining and histology were performed.

Results

No increase in ICP, radiological evidence of ischemia within the MCA territory but without space-occupying edema, and TTC infarct volumes of 7.9+/-5.1% were seen with temporary MCAO. Permanent MCAO resulted in significantly elevated ICP, accompanied by 30% mortality, radiological evidence of space-occupying cerebral edema and TTC infarct volumes of 27.4+/-6.4%.

Conclusions

Permanent proximal MCAO in the sheep results in space-occupying cerebral edema, raised ICP and mortality similar to human malignant MCA stroke. This animal model may prove useful for pre-clinical testing of anti-edema therapies that have shown promise in rodent studies.     

A comparative analysis of the NMR relaxation times of two human colon cancer cell lines

Two human colon cancer cell lines grown in tissue culture were found to have significantly different Nuclear Magnetic Resonance (NMR) relaxation times of water protons in the fresh, intact state and after fragmentation into subcellular fractions. Differences in the protein composition of the subcellular fractions were also demonstrated by protein analysis and gel chromatography. In further studies, these cell lines may be useful to investigate the biochemical basis for the disparity in relaxation times of water protons between tissues which constitutes the basis for Magnetic Resonance Imaging (MRI) tissue contrast.     

Protective effect of resveratrol against oxidative stress in middle cerebral artery occlusion model of stroke in rats

Free radicals have been implicated in neuronal injury during ischemia reperfusion in stroke. Trans resveratrol, a potent antioxidant, polyphenolic compound found in grapes and wines has recently been shown to have neuroprotective activity against oxidative stress in in vitro studies. In the present study the effect of chronic treatment of trans resveratrol was evaluated in focal ischemia induced by middle cerebral artery [MCA] occlusion in rats. Male Wistar rats were pretreated with trans resveratrol 20 mg/kg i.p. for 21 days and were subjected to focal ischemia by occlusion of MCA using intraluminal thread. After two hours of MCA occlusion reperfusion was allowed by retracting the thread. Animals were assessed for motor performance after 24 hours and subsequently rats were sacrificed for estimation of markers of oxidative stress [malondialdehyde [MDA] and reduced glutathione] and for evaluation of volume of infarction. Control group received vehicle and similar protocol was followed. Significant motor impairment, with elevated levels of MDA and reduced glutathione was observed in the vehicle treated MCA occluded rats. Treatment with trans resveratrol prevented motor impairment, rise in levels of MDA and reduced glutathione and also significantly decreased the volume of infarct as compared to control. The study provides first evidence of effectiveness of trans resveratrol in focal ischemia most probably by virtue of its antioxidant property.     

Application of cell penetrating peptide in magnetic resonance imaging of bone marrow mesenchymal stem cells

Tracking the distribution and differentiation of stem cells by high-resolution imaging techniqueswould have significant clinical and research implications.In this study,a model cell-penetrating peptide wasused to carry gadolinium particles for magnetic resonance imaging (MRI) of mesenchymal stem cells (MSCs).MSCs were isolated from rat bone marrow and identified by osteogenic differentiation in vitro.The cell-penetrating peptide labeled with fluorescein-5-isothiocyanate (FITC) and gadolinium was synthesized by asolid-phase peptide synthesis method.Fluorescein imaging analysis confirmed that this new peptide couldinternalize into the cytoplasm and nucleus at room temperature,4℃ and 37℃.Gadolinium were efficientlyinternalized into mesenchymal stem cells by the peptide in a time or concentration-dependent manner,resulting in intercellular shortening of longitudinal relaxation enhancements,which were obviously detectedby 1.5 Tesla Magnetic Resonance Imaging.Cytotoxicity assay and flow cytometric analysis showed thatthe intercellular contrast medium incorporation did not affect cell viability at the tested concentrations.Thein vitro experiment results suggested that the new constructed peptides could be a vector for trackingMSCs.