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A New Rat Model of Thrombotic Focal Cerebral Ischemia
Abstract
We developed a fibrin-rich thrombotic focal cerebral ischemic model with reproducible and predictable infarct volume in rats. In male Wistar rats (n = 77), a thrombus was induced at the origin of the middle cerebral artery (MCA) by injection of thrombin via an intraluminal catheter placed in the intracranial segment of the internal carotid artery (ICA). Thrombus induction and consequent ischemic cell damage were examined by histopathological analysis and neurological deficit scoring, and by measuring changes in cerebral blood flow (CBF) using laser-Doppler flowmetery (LDF), perfusion-weighted imaging (PWI), and by diffusion weighted imaging (DWI). Histopathology revealed that a fibrin-rich thrombus localized to the origin of the right MCA. Regional cerebral blood flow (rCBF) in the right parietal cortex was reduced by 34-58% of preinjection levels after injection of thrombin in rats administered 30 U of thrombin (n = 10). Magnetic resonance imaging (MRI) showed a reduction in CBF and a hyperintensity DWI encompassing the territory supplied by the right MCA. The infarct volume in rats administered 80 U of thrombin was 31.29 +/- 12.9% of the contralateral hemisphere at 24 h (n = 13), and 34.7 +/- 16.4% of the contralateral hemisphere at 168 h (n = 6). Rats administered 30 U of thrombin exhibited a hemispheric infarct volume of 34.0 +/- 14.5% (n = 9) at 24 h and 29.7 +/- 13.9% (n = 8) at 168 h. In addition, thrombotic rats (n = 3) treated with recombinant tissue plasminogen activator (rt-PA) (10 mg/kg) 2 h after thrombosis showed that CBF rapidly returned towards preischemic values as measured by PWI. This model of thrombotic ischemia is relevant to thromboembolic stroke in humans and may be useful in documenting the safety and efficacy of thrombolytic intervention as well as for investigating therapies complementary to antithrombotic therapy.
... The embolic stroke model was modified from a previous study [13]. Rats were anesthetized with an intraperitoneal injection of 6% chloral hydrate (300 mg/kg) with additional injections of 100 mg/kg dose if the depth of anesthesia was reduced. ...
... In some models, thrombin is used as an activator of thrombosis. Blood clots induced by thrombin can be produced in vitro and then injected through a catheter into a cerebral vessel; thus, simulating cardioembolic stroke in humans [8,13]. Alternatively, thrombin can be injected directly into the middle cerebral artery and cause thrombosis in situ [9,10]. ...
... The histological findings were consistent with the impairments in motor and behavioral functions in the same animals 24 h after surgery. Thus, the results of this study (severity and localization of ischemic brain damage) are comparable with the results of similar experimental studies [6,13,27]. However, the relatively high mortality rate in our model can be reduced by improving perioperative laboratory animal care. ...
The thromboembolic ischemia model is one of the most applicable for studying ischemic stroke in humans. The aim of this study was to develop a novel thromboembolic stroke model, allowing, by affordable tools, to reproduce cerebral infarction in rats. In the experimental group, the left common carotid artery, external carotid artery, and pterygopalatine branch of maxillary artery were ligated. A blood clot that was previously formed (during a 20 min period, in a catheter and syringe, by mixing with a thromboplastin solution and CaCl2) was injected into the left internal carotid artery. After 10 min, the catheter was removed, and the incision was sutured. The neurological status of the animals was evaluated using a 20-point scale. Histological examination of brain tissue was performed 6, 24, 72 h, and 6 days post-stroke. All groups showed motor and behavioral disturbances 24 h after surgery, which persisted throughout the study period. A histological examination revealed necrotic foci of varying severity in the cortex and subcortical regions of the ipsilateral hemisphere, for all experimental groups. A decrease in the density of hippocampal pyramidal neurons was revealed. Compared with existing models, the proposed ischemic stroke model significantly reduces surgical time, does not require an expensive operating microscope, and consistently reproduces brain infarction in the area of the middle cerebral artery supply.
... Since over 80% of all human strokes are thromboembolic, a step to better mimic human stroke in animals is the development of embolic stroke models 12 . Embolic models have been used for decades and have been refined over the years to enable research with thrombolytic drugs which are the only successful therapy in phase III clinical trial [13][14][15] . The embolic models are based on the usage of pre-formed blood clots. ...
... This is also obtained in mice but has been lacking in previous embolic models 14 . Previous embolic models used preformed homologous/autologous clot/clots injected into the MCA through a catheter placed in the ICA 15,19,20 . These embolic models have shown high mortality rates and variability in clot placement, infarct lesion size and location 15,35,36 . ...
... Previous embolic models used preformed homologous/autologous clot/clots injected into the MCA through a catheter placed in the ICA 15,19,20 . These embolic models have shown high mortality rates and variability in clot placement, infarct lesion size and location 15,35,36 . The variability of the models may very well resemble the clinical setting; however, the variability also makes the usage of the models in the assessment of new therapeutic strategies arguable. ...
The recent clinical trial (DAWN) suggests that recanalization treatment may be beneficial up to 24 h after stroke onset, thus re-opening avenues for development of new therapeutic strategies. Unfortunately, there is a continuous failure of drugs in clinical trials and one of the major reasons proposed for this translational roadblock is the animal models. Therefore, the purpose of this study was to validate a new thromboembolic stroke rat model that mimics the human pathology, and that can be used for evaluating new strategies to save the brain in conditions compatible with recanalization. Stroke was induced by injection of thrombin into the middle cerebral artery. Recombinant tissue-type plasminogen activator (rt-PA) or saline was administrated at 1 h/4 h after stroke onset, and outcome was evaluated after 24 h. Induced ischemia resulted in reproducible cortical brain injuries causing a decrease in neurological function 24 h after stroke onset. Early rt-PA treatment resulted in recanalization, reduced infarct size and improved neurological functions, while late rt-PA treatment showed no beneficial effects and caused hemorrhagic transformation in 25% of the rats. This validated and established model’s resemblance to human ischemic stroke and high translational potential, makes it an important tool in the development of new therapeutic strategies for stroke.
... Several focal cerebral ischemic stroke models have been developed in a variety of species; these include mechanical occlusion of the middle cerebral artery (MCA) [9][10][11][12] , thromboembolic models [13][14][15] , and photothrombotic models [16] . Global ischemia models, although not formally stroke, are divided into complete and incomplete models of ischemia, which are produced by occluding the cerebral blood fl ow (CBF) completely or incompletely [17] . ...
... Thrombotic focal cerebral ischemia models Zhang et al. modified a rat model of thrombotic focal cerebral ischemia, in which the MCA was selectively occluded by a thrombotic clot made with autologous blood [15,57] . In addition, magnetic resonance imaging (MRI) demonstrated thrombolysis of the occluded MCA by tissue plasminogen activator administration after ischemia in this model. ...
... An embolus was detected in all rats sacrifi ced at 24 h after embolization, and 98% of all injected emboli were lodged at the origin of the MCA. This thromboembolic model accurately mimics the clinical conditions of ischemic stroke, and remains the b est model for research on thrombolytic agents [15,57] . Busch et al. ...
Stroke, including cerebral ischemia, intracerebral hemorrhage, and subarachnoid hemorrhage, is the leading cause of long-term disability and death worldwide. Animal models have greatly contributed to our understanding of the risk factors and the pathophysiology of stroke, as well as the development of therapeutic strategies for its treatment. Further development and investigation of experimental models, however, are needed to elucidate the pathogenesis of stroke and to enhance and expand novel therapeutic targets. In this article, we provide an overview of the characteristics of commonly-used animal models of stroke and focus on the inflammatory responses to cerebral stroke, which may provide insights into a framework for developing effective therapies for stroke in humans.
... A site-specifi c model of embolic focal cerebral ischemia was later developed in rats by injecting homologous clot to MCA via a catheter through external and internal carotid artery (Zhang et al. 1997a), resulting in a reproducible and predictable infarct volume within the territory of MCA (Zhang et al. 1997a). The same research group also established a rat model of thrombotic focal cerebral ischemia (Zhang et al. 1997b) triggered by the injection of thrombin into MCA via an intraluminal catheter. In this model, the cerebral blood fl ow (CBF) rapidly returned to the pre-ischemic levels in animals treated with the recombinant tPA (10 mg/kg) 2 hr after thrombosis (Zhang et al. 1997b). ...
... The same research group also established a rat model of thrombotic focal cerebral ischemia (Zhang et al. 1997b) triggered by the injection of thrombin into MCA via an intraluminal catheter. In this model, the cerebral blood fl ow (CBF) rapidly returned to the pre-ischemic levels in animals treated with the recombinant tPA (10 mg/kg) 2 hr after thrombosis (Zhang et al. 1997b). Similar or improved thrombotic models in rats and mice , Henninger et al. 2006, Dinapoli et al. 2006, Orset et al. 2007, Ren et al. 2012, as well as in large animals, including cynomologus monkey (Kito et al. 2001) and pig (Ringer et al. 2004) were subsequently reported. ...
... A site-specifi c model of embolic focal cerebral ischemia was later developed in rats by injecting homologous clot to MCA via a catheter through external and internal carotid artery (Zhang et al. 1997a), resulting in a reproducible and predictable infarct volume within the territory of MCA (Zhang et al. 1997a). The same research group also established a rat model of thrombotic focal cerebral ischemia (Zhang et al. 1997b) triggered by the injection of thrombin into MCA via an intraluminal catheter. In this model, the cerebral blood fl ow (CBF) rapidly returned to the pre-ischemic levels in animals treated with the recombinant tPA (10 mg/kg) 2 hr after thrombosis (Zhang et al. 1997b). ...
... The same research group also established a rat model of thrombotic focal cerebral ischemia (Zhang et al. 1997b) triggered by the injection of thrombin into MCA via an intraluminal catheter. In this model, the cerebral blood fl ow (CBF) rapidly returned to the pre-ischemic levels in animals treated with the recombinant tPA (10 mg/kg) 2 hr after thrombosis (Zhang et al. 1997b). Similar or improved thrombotic models in rats and mice , Henninger et al. 2006, Dinapoli et al. 2006, Orset et al. 2007, Ren et al. 2012, as well as in large animals, including cynomologus monkey (Kito et al. 2001) and pig (Ringer et al. 2004) were subsequently reported. ...
... Therefore, thromboembolic MCAO is increasingly being used to investigate thrombolytic and neuroprotective agents, as it more closely resembles the pathophysiology of ischemic stroke with varying degrees of tissue reperfusion, thereby providing a better model in which to test thrombolytic therapies. 16,17 The embolic MCAO (eMCAO) model was first reported in rats in 1982, 18 and was further optimized by Overgaard et al. in 1992, 19 and Zhang et al. in 1997. 20 Recently, Zhang et al. 10 developed a standard procedure for focal eMCAO in rats. ...
... 16,17 The embolic MCAO (eMCAO) model was first reported in rats in 1982, 18 and was further optimized by Overgaard et al. in 1992, 19 and Zhang et al. in 1997. 20 Recently, Zhang et al. 10 developed a standard procedure for focal eMCAO in rats. This autologous clot animal model strongly mimics the thromboembolic stroke in humans, but unpredictable recanalization and variation in infarct volume limit its application in studies examining neuroprotective therapies. ...
The embolic middle cerebral artery occlusion (eMCAO) model mimics ischemic stroke due to large vessel occlusion in humans and is amenable to thrombolytic therapy with rtPA. However, two major obstacles, the difficulty of the eMCAO surgery and unpredictable occurrence of clot autolysis, had impeded its application in mice. In this study, we modified catheters to produce suitable fibrin-rich embolus and optimized the eMCAO model using cerebral blood flow (CBF) monitored by both laser Doppler flowmetry (LDF) and 2D laser speckle contrast imaging (LSCI) to confirm occlusion of MCA. The results showed that longer embolus resulted in higher mortality. There was a compensatory increase in MCA territory perfusion after eMCAO associated with decreased infarct volume; however, this was only partly dependent on recanalization as clot autolysis was only observed in ∼30% of mice. Cortical CBF monitoring with LSCI showed that the size of peri-core area at 3 h displayed the best correlation with infarct volume that is attributed to compensatory collateral blood flow. The peri-core area best predicted functional outcome after eMCAO. In summary, we developed a reliable eMCAO mouse model that better mimics embolic ischemic stroke in humans, which will increase the potential for successful translation of stroke neuroprotective therapies.
... Previous studies have shown that different parts of the brain possess different vulnerability factor to ischemic stroke [7,13,63]. Specifically, alterations in the ST, PFC and HC have been linked to the neurological perturbations that closely mimic the behavioral deficits seen in patients suffering from ischemic stroke; as these brain regions play important roles in movement disorders and learning/memory processes [13,64]. Thus, rats exposed to BCCAO showed increased cyto-architectural distortions akin to those seen in the brains of patients with ischemic stroke [7,60]. ...
... Previous studies have shown that different parts of the brain possess different vulnerability factor to ischemic stroke [7,13,63]. Specifically, alterations in the ST, PFC and HC have been linked to the neurological perturbations that closely mimic the behavioral deficits seen in patients suffering from ischemic stroke; as these brain regions play important roles in movement disorders and learning/memory processes [13,64]. Thus, rats exposed to BCCAO showed increased cyto-architectural distortions akin to those seen in the brains of patients with ischemic stroke [7,60]. ...
The effects of Jobelyn® (JB) on neurological deficits and biochemical alterations associated with ischemic stroke induced by bilateral common carotid artery occlusion (BCCAO) in rats were investigated in this study. Male Wistar rats were divided into five groups (n = 8): group 1 served as Sham control; group 2, which served as negative control received normal saline while groups 3–5 were given JB (25, 50 and 100 mg/kg, p.o) daily for 28 days. Then, rats in groups 2–5 were subjected to BCCAO for 30 min and reperfusion afterwards. Neurological deficits were assessed 3 h post-reperfusion using a 9-point neurological scoring scale. The levels of biomarkers of oxidative stress and pro-inflammatory cytokines (tumour necrotic factor-α and interleukin-6), expressions of immunopositive cells of nuclear factor-kappa B (NF-κB) and acetyl-cholinesterase (AChE) activity were determined in brain tissues. Histology of the striatum, prefrontal cortex (PFC) and hippocampus (CA1) was also evaluated. JB improved BCCAO-induced neurological deficits and attenuated increased oxidative stress and AChE activity in rats subjected to BCCAO (p < 0.05). Increased brain levels of tumour necrotic factor-α and interleukin-6 as well as expressions of immunopositive cells of NF-κB were decreased by JB. JB reduced brain damage and also increased population of viable neurons in the striatum, PFC and hippocampus of ischemic stroke rats. These findings suggest that the positive effect of JB on neurological function in rats with ischemic stroke may be related to inhibition of oxidative stress, release of pro-inflammatory cytokines and expressions of immunopositive cells of NF-κB.
... In keeping with our goal to pursue the development of G-CSF as a therapeutic agent to expand the limited therapeutic time window of tPA for ischemic stroke treatment, we conducted a follow-up study to examine whether the drug can reduce delayed tPA treatment-induced HT, worsening of neurological outcomes, and mortality in a thromboembolic (TE) model of stroke, a stringent model that closely mimics the clinical situation of vascular occlusion and reperfusion [14][15][16][17]. We also sought to determine whether G-CSF mobilized EPCs in the context of attenuating delayed tPA-induced HT possibly via vascular repair, and if the therapeutic effects of single G-CSF treatments were sustained at longer post-stroke time points. ...
... Considering that up to 80% of human strokes are caused by thrombosis or embolism [14,15], we used the TE stroke model to examine the efficacy of G-CSF in attenuating delayed tPA-induced HT. Our findings add to the preclinical evidence supporting the potential use of G-CSF to reduce HT associated with delayed tPA therapy [12] and also address a STAIR guideline regarding efficacy assessments of drug candidates in multiple ischemia models prior to clinical development [13]. ...
When given beyond 4.5 h of stroke onset, tissue plasminogen activator (tPA) induces deleterious side effects in the ischemic brain, notably, hemorrhagic transformation (HT). We examined the efficacy of granulocyte-colony stimulating factor (G-CSF) in reducing delayed tPA-induced HT, cerebral infarction, and neurological deficits in a thromboembolic (TE) stroke model, and whether the effects of G-CSF were sustained for longer periods of recovery. After stroke induction, rats were given intravenous saline (control), tPA (10 mg/kg), or G-CSF (300 μg/kg) + tPA 6 h after stroke. We found that G-CSF reduced delayed tPA-associated HT by 47%, decreased infarct volumes by 33%, and improved motor and neurological deficits by 15% and 25%, respectively. It also prevented delayed tPA treatment-induced mortality by 46%. Immunohistochemistry showed 1.5- and 1.8-fold enrichment of the endothelial progenitor cell (EPC) markers CD34+ and VEGFR2 in the ischemic cortex and striatum, respectively, and 1.7- and 2.8-fold increases in the expression of the vasculogenesis marker von Willebrand factor (vWF) in the ischemic cortex and striatum, respectively, in G-CSF-treated rats compared with tPA-treated animals. Flow cytometry revealed increased mobilization of CD34+ cells in the peripheral blood of rats given G-CSF. These results corroborate the efficacy of G-CSF in enhancing the therapeutic time window of tPA for stroke treatment via EPC mobilization and enhancement of vasculogenesis.
... In keeping with our goal to pursue the development of G-CSF as a therapeutic agent to expand the limited therapeutic time window of tPA for ischemic stroke treatment, we conducted a follow-up study to examine whether the drug can reduce delayed tPA treatment-induced HT, worsening of neurological outcomes, and mortality in a thromboembolic (TE) model of stroke, a stringent model that closely mimics the clinical situation of vascular occlusion and reperfusion [14][15][16][17]. We also sought to determine whether G-CSF mobilized EPCs in the context of attenuating delayed tPA-induced HT possibly via vascular repair, and if the therapeutic effects of single G-CSF treatments were sustained at longer post-stroke time points. ...
... Considering that up to 80% of human strokes are caused by thrombosis or embolism [14,15], we used the TE stroke model to examine the efficacy of G-CSF in attenuating delayed tPA-induced HT. Our findings add to the preclinical evidence supporting the potential use of G-CSF to reduce HT associated with delayed tPA therapy [12] and also address a STAIR guideline regarding efficacy assessments of drug candidates in multiple ischemia models prior to clinical development [13]. ...
Abstract: When given beyond 4.5 h of stroke onset, tissue plasminogen activator (tPA) induces deleterious side effects in the ischemic brain, notably, hemorrhagic transformation (HT).We examined the efficacy of granulocyte-colony stimulating factor (G-CSF) in reducing delayed tPA-induced HT, cerebral infarction, and neurological deficits in a thromboembolic (TE) stroke model, and whether the effects of G-CSF were sustained for longer periods of recovery. After stroke induction, rats were given intravenous saline (control), tPA (10 mg/kg), or G-CSF (300 �g/kg) + tPA 6 h after stroke. We found that G-CSF reduced delayed tPA-associated HT by 47%, decreased infarct volumes
by 33%, and improved motor and neurological deficits by 15% and 25%, respectively. It also prevented delayed tPA treatment-induced mortality by 46%. Immunohistochemistry showed 1.5- and 1.8-fold enrichment of the endothelial progenitor cell (EPC) markers CD34+ and VEGFR2 in the ischemic cortex and striatum, respectively, and 1.7- and 2.8-fold increases in the expression of the vasculogenesis marker von Willebrand factor (vWF) in the ischemic cortex and striatum, respectively, in G-CSF-treated rats compared with tPA-treated animals. Flow cytometry revealed increased mobilization of CD34+ cells in the peripheral blood of rats given G-CSF. These results corroborate
the efficacy of G-CSF in enhancing the therapeutic time window of tPA for stroke treatment via EPC mobilization and enhancement of vasculogenesis.
... Although the autologous clot model that mimics thromoboembolic stroke has been developed (Kudo et al. 1982), and efforts have been made to improve its outcome consistency, Zhang et al. 1997b) this model is still not suitable for validating neuroprotective effects because of its uncontrollable reperfusion and unacceptable variation of infarct area Zhang et al. 1997a;Zhang et al. 1997b). Therefore, this model is re-served for clot-related protection mechanisms which other stroke models cannot address. ...
... Although the autologous clot model that mimics thromoboembolic stroke has been developed (Kudo et al. 1982), and efforts have been made to improve its outcome consistency, Zhang et al. 1997b) this model is still not suitable for validating neuroprotective effects because of its uncontrollable reperfusion and unacceptable variation of infarct area Zhang et al. 1997a;Zhang et al. 1997b). Therefore, this model is re-served for clot-related protection mechanisms which other stroke models cannot address. ...
... In this model, a blood clot is formed and introduced into the brain vasculature through the carotid artery or middle cerebral artery. The clot then causes an obstruction in the blood flow, leading to an ischemic stroke [38][39][40]. ...
Despite all current efforts in the field of cerebrovascular disease prevention, stroke remains the leading cause of disability and death worldwide. Animal models are essential tools in stroke research to mitigate its devastating effects. The novelty of this review is the comprehensiveness of the approach and the detailed analysis of progress in the field, translational potential and clinical relevance. Recent research aimed to refine and enhance animal models to better mimic clinical scenario and improve reproducibility. This review provides extensive overview of its classification, underlining their key features, advantages, limitations, and advancements. Additionally, characterization and validation of ischemia models are discussed. Therapeutic strategies such as hypothermia, pharmacological interventions, genetic and molecular approaches, and cell-based therapies are highlighted. This review analyse the challenges and future directions, as well as translational potential and clinical relevance of ischemia models. Incorporating patient-derived cells and genetic modifications in animal models can provide a more personalized and clinically relevant approach. Bridging the gap is challenging and requires careful consideration of multiple factors, including appropriate dose translation, understanding species differences, and aligning protocols. Future studies should focus on optimizing the translational process and conducting well-designed clinical trials to assess the safety and efficacy of promising interventions.
... In the experimental situation, premature recirculation diminished the difference in lesion extent between thrombolytic-treated animals and controls. Unreliable infarctions and variable neurologic deficits can be modified by precise occlusion MCA utilizing microcatheter and LDF.119,120 The main application of the thromboembolic model is to investigate thrombolytic processes. ...
Stroke is a devastating disease with high morbidity and mortality. Animal models are indispensable tools that can mimic stroke processes and can be used for investigating mechanisms and developing novel therapeutic regimens. As a heterogeneous disease with complex pathophysiology, mimicking all aspects of human stroke in one animal model is impossible. Each model has unique strengths and weaknesses. Models such as transient or permanent intraluminal thread occlusion middle cerebral artery occlusion (MCAo) models and thromboembolic models are the most commonly used in simulating human ischemic stroke. The endovascular filament occlusion model is characterized by easy manipulation and accurately controllable reperfusion and is suitable for studying the pathogenesis of focal ischemic stroke and reperfusion injury. Although the reproducibility of the embolic model is poor, it is more convenient for investigating thrombolysis. Rats are the most frequently used animal model for stroke. This review mainly outlines the stroke models of rats and discusses their strengths and shortcomings in detail.
... Since ischemic strokes in humans are mostly caused by thromboembolism [102,103], animal stroke models recapitulating the clinical thromboembolism conditions are considered relevant [104]. These models are useful for assessing neuroprotective activity and re-canalization (thrombolytic) therapy after ischemic stroke [105,106]. In embolic paradigms, emboli (such as thrombotic clots, microspheres and photothrombotic) of different sizes and quantity are injected to interrupt the CBF of targeted arteries in mice, rats, rabbits, pigs, and dogs [107][108][109][110]. ...
Background:
Stroke is a serious neurovascular problem and the leading cause of disability and death worldwide. The disrupted demand to supply ratio of blood and glucose during cerebral ischemia develops hypoxic shock, and subsequently necrotic neuronal death in the affected regions. Multiple causal factors like age, sex, race, genetics, diet, and lifestyle play an important role in the occurrence as well as progression of post-stroke deleterious events. These biological and environmental factors may be contributed to vasculature variable architecture and abnormal neuronal activity. Since recombinant tissue plasminogen activator is the only clinically effective clot bursting drug, there is a huge unmet medical need for newer therapies for the treatment of stroke. Innumerous therapeutic interventions have shown promise in the experimental models of stroke but failed to translate it into clinical counterparts.
Methods:
Original publications regarding pathophysiology, preclinical experimental models, new targets and therapies targeting ischemic stroke have been reviewed since the 1970s.
Results:
We highlighted the critical underlying pathophysiological mechanisms of cerebral stroke and preclinical stroke models. We discuss the strengths and caveats of widely used ischemic stroke models, and commented on the potential translational problems. We also describe the new emerging treatment strategies, including stem cell therapy, neurotrophic factors and gut microbiome-based therapy for the management of post-stroke consequences.
Conclusion:
There are still many inter-linked pathophysiological alterations with regards to stroke, animal models need not necessarily mimic the same conditions of stroke pathology and newer targets and therapies are the need of the hour in stroke research.
... We demonstrated that tPA infusion (10 mg/kg) significantly improved the rCBF compared to saline-treated group. We chose the model of thrombotic ischemia which is relevant to thromboembolic stroke in humans and used to document the safety and efficacy of thrombolytic intervention since it has been introduced (Zhang et al., 1997). The treatment time of tPA varies from 1.5 to 3 hr after ischemia induction to investigate the thrombolytic efficacy of tPA (Asahi et al., 2003;Fan et al., 2017;Zhu et al., 2010). ...
Thrombolytic stroke therapy with tissue plasminogen activator (tPA) is limited by risks of hemorrhagic transformation (HT). We have reported that a new 12/15‐lipoxygenase (12/15‐LOX) inhibitor ML351 reduced tPA related HT in mice subjected to experimental stroke under anticoagulation. In this study, we asked whether ML351 can ameliorate tPA induced HT in an embolic stroke model. Rats were subjected to embolic middle cerebral artery occlusion with 2 or 3 hr ischemia and tPA infusion, with or without ML351. Regional cerebral blood flow was monitored 2 hr after ischemia and continuously monitored for 1 hr after treatment for determining reperfusion. Hemoglobin was determined in brain homogenates and infarct volume was quantified at 24 hr after stroke.12/15‐LOX, cluster of differentiation 68(CD68), immunoglobulin G (IgG), and tight junction proteins expression was detected by immunohistochemistry. ML351 significantly reduced tPA related hemorrhage after stroke without affecting its thrombolytic efficacy. ML351 also reduced blood–brain barrier disruption and improved preservation of junction proteins. ML351 and tPA combination improved neurological deficit of rats even though ML351 did not further reduce the infarct volume compared to tPA alone treated animals. Pro‐inflammatory cytokines were suppressed by ML351 both in vivo and in vitro experiments. We further showed that ML351 suppressed the expression of c‐Jun‐N‐terminal kinase (JNK) in brains and microglia cultures, whereas exogenous 12‐HETE attenuated this effect in vitro. In conclusion, ML351 and tPA combination therapy is beneficial in ameliorating HT after ischemic stroke. This protective effect is probably because of 12/15‐LOX inhibition and suppression of JNK‐mediated microglia/macrophage activation.
image
... Body temperature was maintained at 37°C until recovery from anesthesia. The embolic stroke model was adapted from Zhang et al. [24] and Aoki et al. [25]. Briefly, femoral arterial blood from a donor rat was withdrawn into a PE-50 tube and kept at room temperature for 2 h. ...
Leukotriene B4 (LTB4) has been implicated in ischemic stroke pathology. We examined the prognostic significance of LTB4 levels in patients with acute middle cerebral artery (MCA) infarction and their mechanisms in rat stroke models. In ischemic stroke patients with middle cerebral artery infarction, plasma LTB4 levels were found to increase rapidly, roughly doubling within 24 h when compared to initial post-stroke levels. Further analyses indicate that poor functional recovery is associated with early and more sustained increase in LTB4 rather than the peak levels. Results from studies using a rat embolic stroke model showed increased 5-lipoxygenase (5-LOX) expression in the ipsilateral infarcted cortex compared with sham control or respective contralateral regions at 24 h post-stroke with a concomitant increase in LTB4 levels. In addition, neutrophil influx was also observed in the infarcted cortex. Double immunostaining indicated that neutrophils express 5-LOX and leukotriene A4 hydrolase (LTA4H), highlighting the pivotal contributions of neutrophils as a source of LTB4. Importantly, rise in plasma LTB4 levels corresponded with an increase in LTB4 amount in the infarcted cortex, thereby supporting the use of plasma as a surrogate for brain LTB4 levels. Pre-stroke LTB4 loading increased brain infarct volume in tMCAO rats. Conversely, administration of the 5-LOX-activating protein (FLAP) inhibitor BAY-X1005 or B-leukotriene receptor (BLTR) antagonist LY255283 decreased the infarct volume by a similar extent. To conclude, targeted interruption of the LTB4 pathway might be a viable treatment strategy for acute ischemic stroke.
... The clot was gently injected into the ICA. After 5 min, the catheter was withdrawn [24][25][26]. ...
A rodent model of embolic middle cerebral artery occlusion is used to mimic cerebral embolism in clinical patients. Thrombolytic therapy is the effective treatment for this ischemic injury. However, it is difficult to detect thrombolysis dynamically in living animals. Synchrotron radiation angiography may provide a novel approach to directly monitor the thrombolytic process and assess collateral circulation after embolic stroke. Thirty-six adult Sprague-Dawley rats underwent the embolic stroke model procedure and were then treated with tissue plasminogen activator. The angiographic images were obtained in vivo by synchrotron radiation angiography. Synchrotron radiation angiography confirmed the successful establishment of occlusion and detected the thrombolysis process after the thrombolytic treatment. The time of thrombolytic recanalization was unstable during embolic stroke. The infarct volume increased as the recanalization time was delayed from 2 to 6 h (p < 0.05). The collateral circulation of the internal carotid artery to the ophthalmic artery, the olfactory artery to the ophthalmic artery, and the posterior cerebral artery to the middle cerebral artery opened after embolic stroke and manifested different opening rates (59%, 24%, and 75%, respectively) in the rats. The opening of the collateral circulation from the posterior cerebral artery to the middle cerebral artery alleviated infarction in rats with successful thrombolysis (p < 0.05). The cerebral vessels of the circle of Willis narrowed after thrombolysis (p < 0.05). Synchrotron radiation angiography provided a unique tool to dynamically detect and assess the thrombolysis process and the collateral circulation during thrombolytic therapy.
... The modified BUAL model in rats that we used is one of the reasonable animal models of neonatal HIE (15). Numerous types of animal models have been developed as an HIE model, using various insults, including unilateral common carotid artery occlusion (Vannucci model), bilateral carotid artery occlusion, four-vessel occlusion, middle cerebral occlusion, chronic hypoxia, and cerebrocortical photothrombosis (28,(37)(38)(39)(40)(41). Most of these models used permanent occlusions of arteries in the brain. ...
Brain damage caused by hypoxic ischemic insult during the perinatal period causes hypoxic ischemic encephalopathies (HIEs). Therapeutic hypothermia is indicated for HIE, but since the therapeutic burden is large for its limited therapeutic effectiveness, another strategy is needed. Progesterone (P4) plays a neuroprotective role through the actions of its metabolite, allopregnanolone (Allo), on P4 receptor, GABA (γ-aminobutyric acid)A receptors or both. We examined the therapeutic potential of P4 using a newborn rat model of HIE. Fetal rats were exposed to transient ischemic hypoxia by 30-minute bilateral uterine artery clamping on gestational day 18. After spontaneous birth, newborn pups were subcutaneously injected with P4 (0.10 or 0.01 mg), medroxyprogesterone acetate (MPA, 0.12 mg), or Allo (0.10 mg) through postnatal days (PDs) 1–9. Brain damage in the rats was assessed using the rotarod test at PD50. The HIE insult reduced the rats’ ability in the rotarod task, which was completely reversed by P4 and Allo, but not by MPA. Histological examination revealed that the HIE insult decreased neuronal (the cortex and the hippocampal CA1 region) and oligodendroglial cell density (the corpus callosum) through PD0–50. The axon fiber density and myelin sheath thickness in the corpus callosum were also reduced at PD50. The time-course study revealed that P4 restored oligodendroglial cells by PD5, which was followed by neuroprotective action of P4 that lasted long over the injection period. These results suggest that P4 protects the neonatal brain from HIE insult via restoration of oligodendroglial cells.
... In particular, the characteristics and the impacts of ischemic stroke are multifaceted in nature, in which disrupted cerebral vascular blood flow negatively impacts the neuronal activity and tissue outcome (Zhang and Murphy, 2007;Strong et al., 2007;Nakamura et al., 2010). Moreover, although the effects of ischemic stroke in both patients and experimental animal models are apparent only minutes after blood flow is reduced (Zhang et al., 1997;Hainsworth and Markus, 2008), the progression of ischemia lasts for several days after the initial insult (Hartings et al., 2003;Fabricius et al., 2006), and the functional recovery of the injured brain continues for months and longer (Murphy and Corbett, 2009). While extensive research has been done on the progression of brain injury in focal stroke at the acute phases in small animal models (Zhang and Murphy, 2007;Shin et al., 2006;Jones et al., 2008;Brown et al., 2009), the progression of ischemic conditions and recovery into chronic time scales are understudied (Dirnagl et al., 1999), in large part due to a lack of methods capable of quantifying multiple neurophysiological parameters simultaneously in behaving brains with sufficient spatial resolution over periods of weeks to months. ...
Background:
Despite significant advancements of optical imaging techniques for mapping hemodynamics in small animal models, it remains challenging to combine imaging with spatially resolved electrical recording of individual neurons especially for longitudinal studies. This is largely due to the strong invasiveness to the living brain from the penetrating electrodes and their limited compatibility with longitudinal imaging.
New method:
We implant arrays of ultraflexible nanoelectronic threads (NETs) in mice for neural recording both at the brain surface and intracortically, which maintain great tissue compatibility chronically. By mounting a cranial window atop of the NET arrays that allows for chronic optical access, we establish a multimodal platform that combines spatially resolved electrical recording of neural activity and laser speckle contrast imaging (LSCI) of cerebral blood flow (CBF) for longitudinal studies.
Results:
We induce peri-infarct depolarizations (PIDs) by targeted photothrombosis, and show the ability to detect its occurrence and propagation through spatiotemporal variations in both extracellular potentials and CBF. We also demonstrate chronic tracking of single-unit neural activity and CBF over days after photothrombosis, from which we observe reperfusion and increased firing rates.
Comparison with existing method(s):
This multimodal platform enables simultaneous mapping of neural activity and hemodynamic parameters at the microscale for quantitative, longitudinal comparisons with minimal perturbation to the baseline neurophysiology.
Conclusion:
The ability to spatiotemporally resolve and chronically track CBF and neural electrical activity in the same living brain region has broad applications for studying the interplay between neural and hemodynamic responses in health and in cerebrovascular and neurological pathologies.
... Following ligation and transection of the ECA, the modified PE90 (reducing the optical density to 0.4 mm at one end) with blood clot samples was injected with a 2 ml syringe via the ECA into the ICA. The radioactive intensity of the forelimb cortex (1.0 mm posterior and 5.5 mm lateral to the bregma) was detected using a JC1000-PC Medical Gamma Counter prior to and following embolus injection (9)(10)(11). When the radiation intensity was >2 times greater than the background signal, the model preparation for thromboembolism was considered successful (8). ...
The aim of the present study was to investigate the efficacy of recombinant human prourokinase (rhPro-UK) on thromboembolic stroke in rabbits. A total of 210 rabbits were used in experiments. The 180 thromboembolic stroke rabbits were divided into three therapeutic time windows with six groups in each time window (n=10). The model group was administered saline, the reagent groups were administered rhPro-UK (2.5×, 5× and 10×104 U/kg), and the positive control groups were administered 5×104 urokinase (UK) U/kg and 4.5 mg/kg recombinant human tissue plasminogen activator via intravenous infusion at 3, 4.5 and 6 h after embolism. The remaining 30 rats (that had not undergone occlusion by autologous blood clots) served as a sham group and were administered saline. The radioactive intensity was detected using a medical gamma counter before and after the administration of the drug for 15, 30, 45, 60, 75, 90, 105 and 120 min. At 24 h after treatment, the brain samples were coronally sliced into 5 mm sections and hemorrhage was estimated used a semiquantitative method by counting the number of section faces with hemorrhaging. The plasma was collected for prothrombin time, activated partial thromboplastin time, fibrinogen and thrombin time tests using a solidification method with a blood coagulation factor analyzer. In addition, α2-antiplasmin (α2-AP) was evaluated using ELISA methods using a RT-6100 microplate reader. At the 3 h time point, the thrombolysis rate of rhPro-UK(2.5×, 5× and 10×104 U/kg) was 21.5% (P<0.05), 36.8% (P<0.001) and 55.0% (P<0.001), respectively together with patency rates of 10% (P>0.05), 40% (P<0.05) and 70% (P<0.001). Furthermore, α2-AP levels were reduced by 5.3% (P>0.05), 5.3% (P>0.05) and 18.1% (P<0.05). At the 4.5 h time point, the thrombolysis rate was 18.8% (P<0.05), 29.9% (P<0.01) and 49.0% (P<0.001) together with patency rates of 10% (P>0.05), 30% (P<0.05) and 50% (P<0.01), and α2-AP levels were reduced by 2.4% (P>0.05), 6.5% (P>0.05) and 17.8% (P<0.05). At the 6 h time point, the thrombolysis rate was 14.7% (P<0.05), 24.1%(P<0.01) and 35.7% (P<0.001) together with patency rates of 20% (P>0.05), 30% (P<0.05) and 40% (P<0.01), and α2-AP levels were reduced by 5.7% (P>0.05), 12.7% (P>0.05) and 22.2% (P<0.01). No significant differences (P>0.05) were identified between rhPro-UK (2.5×, 5× and 10×104 U/kg) and the model group regarding hemorrhage type, size and blood coagulation factors at the different time points. Thus, rhPro-UK promoted thrombolysis and recanalization (patency rate), with reduced risk of cerebral hemorrhage, and thus exerted protective effects on cerebral ischemia rabbits.
... For both tests, at least five readings per animal were taken at 15 min intervals, and the average was calculated [15]. Measurement of regional blood flow by laser Doppler flowmetry Regional blood flow in the sciatic nerve was measured at the end of the experiments (8 weeks after the treatment) using laser Doppler flowmetry (LDF PeriFlux PF4, Perimed AB, Järfälla, Sweden) [15,16]. Briefly, under anesthesia (ketamine/xylazine, i.p., 100/10 mg/kg, JHP Pharmaceuticals LLC. MI; LLOYD Inc. Lowa), the mouse was mounted on a Kopf stereotaxic apparatus. ...
We previously demonstrated that treatment of diabetic peripheral neuropathy with the short (4 hours) half-life phosphodiesterase 5 (PDE5) inhibitor, sildenafil, improved functional outcome in diabetic db/db mice. To further examine the effect of PDE5 inhibition on diabetic peripheral neuropathy, we investigated the effect of another potent PDE5 inhibitor, tadalafil, on diabetic peripheral neuropathy. Tadalafil is pharmacokinetically distinct from sildenafil and has a longer half-life (17+hours) than sildenafil. Diabetic mice (BKS.Cg-m+/+Leprdb/J, db/db) at age 20 weeks were treated with tadalafil every 48 hours for 8 consecutive weeks. Compared with diabetic mice treated with saline, tadalafil treatment significantly improved motor and sensory conduction velocities in the sciatic nerve and peripheral thermal sensitivity. Tadalafil treatment also markedly increased local blood flow and the density of FITC-dextran perfused vessels in the sciatic nerve concomitantly with increased intraepidermal nerve fiber density. Moreover, tadalafil reversed the diabetes-induced reductions of axon diameter and myelin thickness and reversed the diabetes-induced increased g-ratio in the sciatic nerve. Furthermore, tadalafil enhanced diabetes-reduced nerve growth factor (NGF) and platelet-derived growth factor-C (PDGF-C) protein levels in diabetic sciatic nerve tissue. The present study demonstrates that tadalafil increases regional blood flow in the sciatic nerve tissue, which may contribute to the improvement of peripheral nerve function and the amelioration of diabetic peripheral neuropathy.
... Behavior. Animal behavior was evaluated 24 h, 96 h, and 168 h after CI-RP based on a modified Bederson scoring method [16]. When a rat's tail was lifted, if the bilateral forelimbs were symmetrically stretched, the rat was given a score of 0. If wrist bending, elbow bending, shoulder internal rotation, or both wrist/elbow bending and shoulder internal rotation of the left forelimb were observed, the rat was given a score of 1, 2, 3, or 4, respectively. ...
The mechanisms by which Shaoyao-Gancao decoction (SGD) inhibits the production of inflammatory cytokines in serum and brain tissue after cerebral ischemia-reperfusion (CI-RP) in rats were investigated. A right middle cerebral artery occlusion was used to induce CI-RP after which the rats were divided into model (
n
=
39
), SGD (
n
=
28
), clopidogrel (
n
=
25
) and sham operated (
n
=
34
) groups. The Bederson scale was used to evaluate changes in behavioral indices. The levels of IL-1
β
, TNF-
α
, MCP-1, IL-10, RANTES, VEGF, and TGF-
β
1 in the serum and infarcted brain tissues were measured. Nissl body and immunohistochemical staining methods were used to detect biochemical changes in neurons, microglial cells, and astrocytes. Serum levels of VEGF, TNF-
α
, MCP-1, IL-1
β
, and IL-10 increased significantly 24 h after CI-RP. In brain tissue, levels of TNF-
α
and IL-1
β
significantly increased 24 h after CI-RP, whereas levels of TGF-
β
1 and MCP-1 were significantly higher 96 h after CI-RP (
P
<
0.05
). SGD or clopidogrel after CI-RP reduced TNF-
α
and IL-1
β
levels in brain tissue and serum levels of MCP-1, IL-1
β
, and IL-10. SGD increased the number of NeuN-positive cells in infarcted brain tissue and reduced the number of IBA1-positive and GFAP-positive cells. The efficacy of SGD was significantly higher than that of clopidogrel.
At present, due to the rapid progress of treatment technology in the acute phase of ischaemic stroke, the mortality of patients has been greatly reduced but the number of disabled survivors is increasing, and most of them are elderly patients. Physicians and rehabilitation therapists pay attention to develop all kinds of therapist techniques including physical therapy techniques, robot-assisted technology and artificial intelligence technology, and study the molecular, cellular or synergistic mechanisms of rehabilitation therapies to promote the effect of rehabilitation therapy. Here, we discussed different animal and in vitro models of ischaemic stroke for rehabilitation studies; the compound concept and technology of neurological rehabilitation; all kinds of biological mechanisms of physical therapy; the significance, assessment and efficacy of neurological rehabilitation; the application of brain–computer interface, rehabilitation robotic and non-invasive brain stimulation technology in stroke rehabilitation.
In most preclinical models of focal ischemic stroke, vascular occlusion is performed under general anesthesia. However, anesthetic agents exert confounding effects on mean arterial blood pressure (MABP), cerebrovascular tone, oxygen demand, and neurotransmitter receptor transduction. Moreover, the majority of studies do not use a blood clot, which more fully models embolic stroke. Here, we developed a blood clot injection model to produce large cerebral artery ischemia in unanesthetized rats. Under isoflurane anesthesia, an indwelling catheter was implanted in the internal carotid artery via a common carotid arteriotomy and preloaded with a 0.38-mm-diameter clot of 1.5, 3, or 6 cm length. After discontinuing anesthesia, the rat was returned to a home cage where it regained normal mobility, grooming, eating activity, and a stable recovery of MABP. One hour later, the clot was injected over a 10-s period and the rats were observed for 24 h. Clot injection produced a brief period of irritability, then 15-20 min of complete inactivity, followed by lethargic activity at 20-40 min, ipsilateral deviation of the head and neck at 1-2 h, and limb weakness and circling at 2-4 h. Neurologic deficits, elevated MABP, infarct volume, and increased hemisphere water content varied directly with clot size. Mortality after 6-cm clot injection (53%) was greater than that after 1.5-cm (10%) or 3-cm (20%) injection. Combined non-survivor groups had the greatest MABP, infarct volume, and water content. Among all groups, the pressor response correlated with infarct volume. The coefficient of variation of infarct volume with the 3-cm clot was less than that in published studies with the filament or standard clot models, and therefore may provide stronger statistical power for stroke translational studies. The more severe outcomes from the 6-cm clot model may be useful for the study of malignant stroke.
Ischemic stroke (IS) is one of the most common causes of disability and death. Thrombolysis and neuroprotection are two current major therapeutic strategies to overcome the ischemic and reperfusion damages. Here, we have designed a novel peptide-templated MnO2 nanozyme (PNzyme/MnO2 ) that integrates the thrombolytic activity of the functional peptides with the ROS scavenging ability of the nanozyme. Through the self-assembled polypeptides that contains multiple functional motifs, the novel peptide-templated nanozyme is able to bind fibrin in the thrombus, cross blood brain barrier, and finally accumulate in the ischemic neuronal tissues, where the thrombolytic motif is "switch-on" by the action of thrombin. In mice and rat IS models, the PNzyme/MnO2 prolonged blood circulation time and exhibited strong thrombolytic action, and reduced the ischemic damages in brain tissues. Moreover, this peptide-templated nanozyme also effectively inhibited the activation of astrocytes and the secretion of pro-inflammatory cytokines. These data indicated that rationally designed PNzyme/MnO2 nanozyme exerts both thrombolytic and neuroprotective actions. Giving its long half-life in the blood and ability to target brain thrombi, the biocompatible nanozyme may serve as a novel therapeutic agent to improve the efficacy and prevent secondary thrombosis during the treatment of IS. This article is protected by copyright. All rights reserved.
Occlusions in the blood vessels caused by blood clots, referred to as thrombosis, and the subsequent outcomes are leading causes of morbidity and mortality worldwide. In vitro and in vivo models of thrombosis have advanced our understanding of the complex pathways involved in its development and allowed the evaluation of different therapeutic approaches for its management. This review summarizes different commonly used approaches to induce thrombosis in vivo and in vitro, without detailing the protocols for each technique or the mechanism of thrombus development. For ease of flow, a schematic illustration of the models mentioned in the review is shown below. Considering the number of available approaches, we emphasize the importance of standardizing thrombosis models in research per study aim and application, as different pathophysiological mechanisms are involved in each model, and they exert varying responses to the same carried tests. For the time being, the selection of the appropriate model depends on several factors, including the available settings and research facilities, the aim of the research and its application, and the researchers’ experience and ability to perform surgical interventions if needed.
In recent years, medical developments have resulted in an increase in human life expectancy. Some developed countries now have a larger population of individuals aged over 64 than those under 14. One consequence of the ageing population is a higher incidence of certain neurodegenerative disorders. In order to prevent these, we need to learn more about them. This book provides up-to-date information on the use of transgenic mouse models in the study of neurodegenerative disorders such as Alzheimer's and Huntington's disease. By reproducing some of the pathological aspects of the diseases, these studies could reveal the mechanism for their onset or development. Some of the transgenic mice can also be used as targets for testing new compounds with the potential to prevent or combat these disorders. The editors have extensive knowledge and experience in this field and the book is aimed at undergraduates, postgraduates and academics. The chapters cover disorders including: Alzheimer's disease, Parkinson's disease, Huntington's and other CAG diseases, amyotrophic lateral sclerosis (ALS), recessive ataxias, disease caused by prions, and ischemia.
Cerebral ischemic stroke is a leading cause of death and disability, but current pharmacological therapies are limited in their utility and effectiveness. In vitro and in vivo models of ischemic stroke have been developed which allow us to further elucidate the pathophysiological mechanisms of injury and investigate potential drug targets. In vitro models permit mechanistic investigation of the biochemical and molecular mechanisms of injury but are reductionist and do not mimic the complexity of clinical stroke. In vivo models of ischemic stroke directly replicate the reduction in blood flow and the resulting impact on nervous tissue. The most frequently used in vivo model of ischemic stroke is the intraluminal suture middle cerebral artery occlusion (iMCAO) model, which has been fundamental in revealing various aspects of stroke pathology. However, the iMCAO model produces lesion volumes with large standard deviations even though rigid surgical and data collection protocols are followed. There is a need to refine the MCAO model to reduce variability in the standard outcome measure of lesion volume. The typical approach to produce vessel occlusion is to induce an obstruction at the origin of the middle cerebral artery and reperfusion is reliant on the Circle of Willis (CoW). However, in rodents the CoW is anatomically highly variable which could account for variations in lesion volume. Thus, we developed a refined approach whereby reliance on the CoW for reperfusion was removed. This approach improved reperfusion to the ischemic hemisphere, reduced variability in lesion volume by 30%, and reduced group sizes required to determine an effective treatment response by almost 40%. This refinement involves a methodological adaptation of the original surgical approach which we have shared with the scientific community via publication of a visualised methods article and providing hands-on training to other experimental stroke researchers.
Cerebral ischemic stroke is a leading cause of death and disability, but current pharmacological therapies are limited in their utility and effectiveness. In vitro and in vivo models of ischemic stroke have been developed which allow us to further elucidate the pathophysiological mechanisms of injury and investigate potential drug targets. In vitro models permit mechanistic investigation of the biochemical and molecular mechanisms of injury but are reductionist and do not mimic the complexity of clinical stroke. In vivo models of ischemic stroke directly replicate the reduction in blood flow and the resulting impact on nervous tissue. The most frequently used in vivo model of ischemic stroke is the intraluminal suture middle cerebral artery occlusion (iMCAO) model, which has been fundamental in revealing various aspects of stroke pathology. However, the iMCAO model produces lesion volumes with large standard deviations even though rigid surgical and data collection protocols are followed. There is a need to refine the MCAO model to reduce variability in the standard outcome measure of lesion volume. The typical approach to produce vessel occlusion is to induce an obstruction at the origin of the middle cerebral artery and reperfusion is reliant on the Circle of Willis (CoW). However, in rodents the CoW is anatomically highly variable which could account for variations in lesion volume. Thus, we developed a refined approach whereby reliance on the CoW for reperfusion was removed. This approach improved reperfusion to the ischemic hemisphere, reduced variability in lesion volume by 30%, and reduced group sizes required to determine an effective treatment response by almost 40%. This refinement involves a methodological adaptation of the original surgical approach which we have shared with the scientific community via publication of a visualised methods article and providing hands-on training to other experimental stroke researchers.
The work in this study has been mainly directed towards developing consistent protocols for an animal model of ischaemic stroke, suitable for novel drug combination strategies, using the intraluminal suture method of middle cerebral artery occlusion (MCAO) in the rat. Following optimisation of the measurement protocols, it was found that more consistent volumes of cerebral infarction were produced in the animal model when a silicone-coated type of intraluminal suture was used for permanent MCAO in Sprague-Dawley rats (coefficient of variation (cv) 19.9%), compared to an uncoated type (cv 66.6%). This more consistent protocol was used in a therapeutic screening study, combining 619C89, a sodium channel blocker to inhibit glutamate release, with BB-823, a platelet activating factor (PAF) antagonist to inhibit inflammatory responses. Although both agents individually reduced infarct volume, combined administration was only slightly and not significantly more effective. This may have been due to either a ceiling effect or the possibility that both agents targetted the same pathological mechanism. Both glutamate and PAF have been previously shown to induce the expression of cyclo-oxygenase (COX-2). However, molecular studies showed that although COX-2 induction was glutamate-mediated, 619C89 had no effect on the process, and the effect of BB-823 was only slight. A persistent problem with the model was the degree of residual variability, which was possibly obscuring small but significant differences. An attempt was made to improve reproducibility of the protocol further, by investigating the effect of animal strain. In the context of permanent MCAO, Fischer-344 rats had more consistent infarct volumes (cv 10%). Extending this approach to the context of temporary MCAO, Wistar rats had more consistent results (cv 9.5%). The direction of this study turned from investigating novel therapies to developing consistent protocols. Since reliable animal models are essential for preclinical studies, the present findings should significantly benefit stroke research.
Despite recent advances in recanalization therapy, mechanical thrombectomy will never be a treatment for every ischemic stroke because access to mechanical thrombectomy is still limited in many countries. Moreover, many ischemic strokes are caused by occlusion of cerebral arteries that cannot be reached by intra-arterial catheters. Reperfusion using thrombolytic agents will therefore remain an important therapy for hyperacute ischemic stroke. However, thrombolytic drugs have shown limited efficacy and notable hemorrhagic complication rates, leaving room for improvement. A comprehensive understanding of basic and clinical research pipelines as well as the current status of thrombolytic therapy will help facilitate the development of new thrombolytics. Compared with alteplase, an ideal thrombolytic agent is expected to provide faster reperfusion in more patients; prevent re-occlusions; have higher fibrin specificity for selective activation of clot-bound plasminogen to decrease bleeding complications; be retained in the blood for a longer time to minimize dosage and allow administration as a single bolus; be more resistant to inhibitors; and be less antigenic for repetitive usage. Here, we review the currently available thrombolytics, strategies for the development of new clot-dissolving substances, and the assessment of thrombolytic efficacies in vitro and in vivo .
Vascular cognitive impairment (VCI) refers to the entire spectrum of vascular brain pathologies that contribute to cognitive deficits, ranging from subjective cognitive decline to dementia. The main pathologies in VCI are infarcts and white matter hyperintensities due to ischemia. VCI rodent models can be divided into surgical models (e.g., MCAO, BCAO, BCAS, 2-VO, 4-VO) and genetic models (e.g., SHR/SP, T2DM, CAA, CADASIL) based on construction methods. However, no single model can fully recapitulate the pathogenesis of VCI, and choosing the appropriate model for different research purposes would be of crucial importance. Here, we have summarized the commonly used rodent VCI models and discussed their advantages and limitations to provide a necessary reference for selecting suitable animal models to investigate the molecular pathways involved in VCI and develop therapeutic interventions.
Schwann cell-derived exosomes communicate with dorsal root ganglia (DRG) neurons. The present study investigated the therapeutic effect of exosomes derived from healthy Schwann cells (SC-Exos) on diabetic peripheral neuropathy (DPN). We found that intravenous administration of SC-Exos to type II diabetic db/db mice with peripheral neuropathy remarkably ameliorated DPN by improving sciatic nerve conduction velocity and increasing thermal and mechanical sensitivity. These functional improvements were associated with the augmentation of epidermal nerve fibers, and remyelination of sciatic nerves. Quantitative RT-PCR and Western blot analysis of sciatic nerve tissues showed that the SC-Exo treatment reversed diabetes-reduced microRNA (miR)-21, -27a and -146a and diabetes-increased Semaphorin 6A (SEMA6A), Ras homolog gene family, member A (RhoA), phosphatase and tensin homolog (PTEN), and nuclear factor-κB (NF-κB). In vitro data showed that SC-Exos promoted neurite outgrowth of diabetic DRG neurons and migration of Schwann cells challenged by high glucose. Collectively, these novel data provide evidence that SC-Exos have a therapeutic effect on DPN in mice and suggest that SC-Exos modulation of miRs contribute to this therapy.
Multiple studies on cerebral ischemia have been performed in animal models to propose different strategies of neuroprotection that mitigate either the early or late consequences of the disease. These therapies have been successful in reducing the volume of infarction, the proinflammatory cascade, and the amount of free radicals, as well as reversing markers of neurodegeneration, among other events. However, when those strategies are translated to clinical studies, their effectiveness is not reproduced. This review will focus on highlighting some of the main limitations of the animal models of stroke that lead to unsuccessful translational therapies and the common risk factors in humans that should be carefully considered in the experimental design of future studies to generate a more realistic spatiotemporal physiopathology and improve therapeutic efficacy in cerebral ischemia.
We discuss two complementary methods for the study of cerebral blood flow and brain function in response to the occlusion of individual, targeted blood vessels. These bear on the study of microstroke and vascular dysfunction in cortex. One method makes use of linear optical absorption by a photosensitizer, transiently circulated in the blood stream, to induce an occlusion in a surface or near-surface vessel. The second method makes use of nonlinear optical interactions, without the need to introduce an exogenous absorber, to induce an occlusion in a subsurface microvessel. A feature of both methods is that the dynamics of blood flow and functional aspects of the vasculature and underlying neurons in the neighborhood of the occluded vessel may be monitored before, during, and after the occlusion. We present details of both methods and associated surgical procedures, along with example data from published studies.
Autophagy is an evolutionarily conserved process of cellular self-degradation and recycling of redundant cytoplasmic entities by lysosomal enzymes. Moreover, autophagy also plays critical roles in controlling several biochemical and molecular neuronal physiology such as growth, survival and metabolism. The autophagy process constantly occurs at basal level under normal physiological conditions and gets increased during stress conditions such as starvation and hypoxia. In neuronal cells, it is a vital homeostasis mechanism that helps in the maintenance of protein quality control. In various neurological disorders, several crucial pro-survival and anti-apoptotic effects of autophagy have been reported. However, the function of autophagy in ischaemic stroke (IS) is highly controversial and still debated. Some reports show that it protects neurons during IS, while others advocate it to be neurodegenerative. Thus, the present chapter deals with the possible function of autophagy in ischaemic stroke along with the discussion of various factors influencing the action of autophagy in ischaemic stroke.
Introduction and aim of the study. Ischemic stroke develops in conjunction with interruption of blood flow in microvessels that depends on rheological blood properties. There is a lack of knowledge in hemorheological features of experimental stroke making more difficult to value the relevance of stroke models. The study aims investigation of microhemorheological parameters in two experimental stroke models - thromboembolic model and middle cerebral artery (MCA) ligation model. Methods. Male Wistar rats were subjected to focal brain ischemia in MCA ligation stroke model or thromboembolic stroke model. The neurological deficit, the size of ischemic brain lesion and hemorheological parameters (hematocrit, kinetics of red blood cells (RBC) (dis)aggregation and RBC deformability) were evaluated. Results. The neurological deficit was correlated with the size of brain ischemic lesion. The increased rate of RBC aggregate formation was detected in both stroke models. At the same time, the strength of RBC aggregates changed in a model-dependent manner, namely, it raised sharply in the MCA ligation stroke model, but was somewhat decreased in thromboembolic stroke model. Conclusion. The focal stroke models produce repeatable and neurologically significant lesions of brain followed with fundamental changes in the hemorheological parameters. The way of ischemia producing can be crucial for the direction of hemorheological changes.
Background:
Injection of a clot into the internal carotid artery is an experimental model of ischemic stroke that is considered to closely mimic embolic stroke in humans. In this model, the common carotid artery typically remains temporarily occluded to permit time for stabilization of the clot in the middle cerebral artery. However, the associated lengthening of the anesthesia duration could affect arterial blood pressure and stroke outcome.
New method:
We refined the model by examining how increasing isoflurane anesthesia duration from 30 to 60 min after clot embolization affects mortality, infarct volume, edema, blood-brain barrier permeability, and the 8-h post-ischemic time course of blood pressure, which has not been reported previously in this model.
Results:
We found that arterial pressure increased after discontinuing anesthesia in both embolized groups and that the increase was greater than in the corresponding non-embolized sham-operated rats. At 24 h, the group with 60-min post-ischemia anesthesia exhibited greater brain water content and a greater ipsilateral-to-contralateral ratio of extravasated Evans blue dye. Mortality was greater in the 60-min group, but infarct volume among survivors was not different from that in the 30-min anesthesia group.
Comparison with existing methods:
This study refines the embolic stroke model by demonstrating the importance of minimizing the duration of anesthesia after embolization.
Conclusions:
These data indicate that early discontinuation of isoflurane anesthesia after clot embolization permits an earlier hypertensive response that limits edema formation and mortality without significantly affecting infarct volume in survivors, thereby decreasing the required number of animals.
Developing reliable and reproducible animal model is of great importance in the therapeutic research of ischemic stroke. The location and volume of injury are varied in different animal models. Researchers choose different animal models according to the research purposes. In this chapter, we summarized the system of ischemic stroke models.
We evaluated the efficacy and safety of human recombinant prourokinase ( rhpro-UK) on thromboembolic stroke in rats. 60 rats with thromboembolic stroke were divided into 6 groups(n=10). The model group was given saline, the reagent groups were given rhpro-UK (5, 10, 20×10(4) U/kg), and positive control groups were given urokinase (UK) 10×10(4) U/kg and recombinant tissue plasminogen activator (rt-PA) 9mg/kg through intravenous infusion at 1.5h after embolism. And other 10 rats without occluded by autologous blood clots as the sham group were given saline. At 6h after treatment, neurological deficit score and Magnetic Resonance Imaging(MRI) including SE-T2WI and DWI sequence scanning were measured. At 24h after treatment, the brain was cut for 2,3,5-triphenyltetrazolium chloride (TTC) staining and aspectrophotometric assay to measure the infarct area and intracerebral hemorrhage after neurological deficit detection. rhpro-UK(5, 10, 20×10(4) U/kg) improved neurological disorder by 39.1±19.7%(n=10, P>0.05), 65.2±14.2%(n=10,P<0.01) and 65.2±14.2%(n=10,P<0.01) maximally; decreased brain lesion volume by 36.7±34.8%(n=10,P<0.05), 77.6±7.7%(n=10,P<0.01) and 80.5±6.9%(n=10,P<0.01); decreased infarction area by 338.2±24.0%(n=10,P<0.01), 73.9±5.2%(n=10,P<0.001) and 79.7±4.0%(n=10,P<0.001) respectively, and there were no statistics difference between rhpro-UK(5, 10, 20×10(4) U/kg) and each positive groups at intracerebral hemorrhage(P>0.05). Rhpro-UK improved the damaged neural function, decreased the extent of the disease and did not raise bleeding, had protective effects for cerebral ischemia in rats.
Objective: This study aimed to introduce a modified animal model of middle cerebral arterial occlusion (MCAO) through placement of intraluminal spindle-shaped head suture by comparing the traditional MCAO model.
Methods: A total of 60 male Spraque-Dawley (SD) rats were divided into two groups and MCAO was induced using spindle-shaped head suture or round head suture. The mortality, infarct volume, neurological function, success rate of the surgery, and stability of modeling were examined to evaluate the effectiveness of this model.
Results: Our results showed the success rate was 90.0% in spindle-shaped head group and 83.3% in round head group showing no significant difference; spindle-shaped head achieved a better establishment of MCAO model as shown in neurological examination. The infarct volume was 31.99 ± 5.44% in spindle-shaped head group and was significantly higher than in round head group (24.59 ± 7.17%; p < 0.05), and the coefficient of variation of infarct volume in spindle-shaped head group was lower than in round head group.
Conclusion: Our findings indicate that the modified suture induces a more reproducible and stable ischemic stroke following MCAO in SD rats.
Cerebral ischemia in diabetics results in severe brain damage. Different animal models of cerebral ischemia have been used to study the aggravation of ischemic brain damage in the diabetic condition. Since different disease conditions such as diabetes differently affect outcome following cerebral ischemia, the Stroke Therapy Academic Industry Roundtable (STAIR) guidelines recommends use of diseased animals for evaluating neuroprotective therapies targeted to reduce cerebral ischemic damage. The goal of this review is to discuss the technicalities and pros/cons of various animal models of cerebral ischemia currently being employed to study diabetes-related ischemic brain damage. The rational use of such animal systems in studying the disease condition may better help evaluate novel therapeutic approaches for diabetes related exacerbation of ischemic brain damage.
This Review describes the current status of poststroke epilepsy (PSE) with an emphasis on poststroke epileptogenesis modeling for testing new therapeutic agents. Stroke is a leading cause of epilepsy in an aging population. Late-onset “epileptic” seizures have been reported in up to 30% cases after stroke. Nevertheless, the overall prevalence of PSE is 2–4%. Rodent models of stroke have contributed to our understanding of the relationship between seizures and the underlying ischemic damage to neurons. To understand whether acutely generated stroke events lead to a chronic phenotype more closely resembling PSE with recurrent seizures, a limited variety of approaches emerged in early 2000s. These limited methods of causing an occlusion in mice and rats show different infarct size and neurological deficits. The most often employed procedure for inducing focal ischemia is the middle cerebral artery occlusion. This mimics the pathophysiology seen in humans in terms of extent of damage to cortex and striatum. Photothrombosis and endothelin-1 models can similarly evoke episodes of ischemic stroke. These models are well suited to studying mechanisms and biomarkers of epileptogenesis or optimizing novel drug discoveries. However, modeling of PSE is tedious, is highly variable, and lacks validity; therefore, it is not widely implemented in epilepsy research. Moreover, the relevance of ischemic models to specific forms of human stroke remains unclear. Stroke modeling in young male rodents lacks clinical relevance to elderly populations and especially to women, likely as a result of sex differences. Nevertheless, because of the neuronal damage and epileptogenic insult that these models trigger, they are helpful tools in studying acquired epilepsy and prophylactic drug therapy.
Acute ischemic stroke, in which a clot of blood manages to obstruct an artery supplying blood to a part of the brain and leads to brain damage, ranks as the third leading cause of death and is the leading cause of serious long-term disability in the United States. At present, the administration of the clot-dissolving drug, tissue-plasminogen activator, remains the only FDA-approved reperfusion therapy for acute ischemic stroke. Unfortunately, relatively few people (4–14%, depending on the study) access medical care in time or are eligible enough for treatment to benefit [8,10,24]. There is a need to develop additional therapies. The occlusion that causes a stroke is a dynamic one that is prone to spontaneous re-canalization and varying degrees of spontaneous reperfusion. The incorporation of this tendency into a translational stroke model will maximize clinical relevance.
This chapter aims at summarizing current knowledge on experimental systems for analyzing the role of white matter injury relevant to stroke. In this chapter, we will provide a broad but brief survey of existing models at the cell, tissue, and whole-animal levels. Experimental approaches have recently allowed a better understanding of the molecular and cellular pathways underlying oligodendrocyte and oligodendrocyte precursor cell damage and demyelination. Since white matter damage is a clinically important part of stroke, a systematic utilization of these cell/tissue/whole-animal platforms related to white matter pathophysiology may eventually lead us to discover new targets for treating stroke.
Background:
Short wave (SW), a pattern of electromagnetic therapy, achieves an oscillating electromagnetic field. It has been reported that it may have a potential effect on cerebral injury. The present study was designed to investigate the potential role and possible mechanism of SW in focal cerebral ischemia/reperfusion (I/R) injury in rats. Secretory pathway Ca(2+)/Mn(2+) ATPase isoform 1 is a major component of Golgi apparatus stress. It has been reported as representative of Golgi apparatus stress.
Methods:
Up to 120 minutes of middle cerebral artery occlusion (MCAO) and reperfusion injury was induced in male Sprague-Dawley rats. Different sessions of SW daily were administered over head after reperfusion from day 1 to day 7. Functional recovery scores, survival rates, infarct volume analysis, electron microscope test, and western blotting studies were used to analyze the therapy.
Results:
SW protected against neuronal death and apoptosis in cornu ammon 1 region of hippocampus by reducing neuronal deficit, infarct volume, and ultrastructure. SW partly inhibited upregulation of caspase3. In addition, the expression of secretory pathway Ca(2+)-ATPase isoform 1 (SPCA1) was upregulated by SW.
Conclusions:
Our data indicate that SW can be protected against focal cerebral I/R injury, and the influence on Golgi apparatus stress might provide us a new perspective in further study. To the authors' knowledge, this is the first report using SW to increase expression of SPCA1 indicating modulate Golgi apparatus stress in MCAO and reperfusion model.
Background and purpose:
The debate over the fact that experimental drugs proposed for the treatment of stroke fail in the translation to the clinical situation has attracted considerable attention in the literature. In this context, we present a retrospective pooled analysis of a large data set from preclinical studies, to examine the effects of early versus late administration of intravenous recombinant tissue-type plasminogen activator.
Methods:
We collected data from 26 individual studies from 9 international centers (13 researchers; 716 animals) that compared recombinant tissue-type plasminogen activator with controls, in a unique mouse model of thromboembolic stroke induced by an in situ injection of thrombin into the middle cerebral artery. Studies were classified into early (<3 hours) versus late (≥3 hours) drug administration. Final infarct volumes, assessed by histology or magnetic resonance imaging, were compared in each study, and the absolute differences were pooled in a random-effect meta-analysis. The influence of time of administration was tested.
Results:
When compared with saline controls, early recombinant tissue-type plasminogen activator administration was associated with a significant benefit (absolute difference, -6.63 mm(3); 95% confidence interval, -9.08 to -4.17; I(2)=76%), whereas late recombinant tissue-type plasminogen activator treatment showed a deleterious effect (+5.06 mm(3); 95% confidence interval, +2.78 to +7.34; I(2)=42%; Pint<0.00001). Results remained unchanged after subgroup analyses.
Conclusions:
Our results provide the basis needed for the design of future preclinical studies on recanalization therapies using this model of thromboembolic stroke in mice. The power analysis reveals that a multicenter trial would require 123 animals per group instead of 40 for a single-center trial.
Abstract
In patients affected with stroke, most ischemic episodes (80-90%)
occur due to occlusion of middle cerebral artery (MCA).
Some of blood through femoral artery was withdrawn into a PE- 50
catheter and kept for 2 h in room temperature and 22 h at 4°C to allow
old clot formation. Then the catheter was advanced 17 mm in the
internal carotid artery until its tip was 1 to 2 mm away from MCA
origin. 3-5 Yl the preformed clot or 5 Yl saline (for sham- operated
animals) were injected. Behavioral deficits and seizure activities were
recorded at 2, 24, and 48 hours after clot injection. Then the rats were
decapitated to remove the brains and prepare them for Tetrazoliume
chloride (TTC) staining and analyzing.
When 3 or 5 Yl clots were injected, infarct volume was 29.35±1.26%
and 16.15±94% respectively (p<0.05). Brain edema also was
8.6±1.65% and 3±1.74%, respectively (p<0.05). There was a
significant correlation between infarct volume and brain edema
(r=0.56, p<0.05). Behavioral deficit score at 48 hours after clot
injection between two groups were significantly different (p<0.05).
This model is very similar to thromboembolic stroke in human and
prepares a reliable method for investigating the stroke mechanism. It
would also be useful for studying thromboembolic agents' effect on
stroke and brain ischemic injury.
An accurate, reproducible method for determining the infarct volumes of gray matter structures is presented for use with presently available image analysis systems. Areas of stained sections with optical densities above that of a threshold value are automatically recognized and measured. This eliminates the potential error and bias inherent in manually delineating infarcted regions. Moreover, the volume of surviving normal gray matter is determined rather than that of the infarct. This approach minimizes the error that is introduced by edema, which distorts and enlarges the infarcted tissue and surrounding white matter.Keywords: Brain; Infarction; Nissl stain; Succinate dehydrogenase
Thrombin-induced expression of endothelial adhesivity toward neutrophils (PMN) was studied using human umbilical vein endothelial cells (HUVEC). HUVEC were challenged with human alpha-thrombin for varying durations up to 120 min, after which the cells were fixed with 1% paraformaldehyde and 51Cr-labeled human PMN were added to determine PMN adhesion. Endothelial adhesivity increased within 15 min after alpha-thrombin exposure, and the response persisted up to 120 min. Expression of endothelial adhesion proteins, P-selectin (GMP-140, PADGEM, CD62), and intercellular adhesion molecule-1 (ICAM-1; CD54) on the endothelial surface was quantitated by increase in the specific binding of anti-P-selectin mAb G1 and anti-ICAM-1 mAb RR1/1 labeled with 125I. P-selectin expression was maximal at 5-15 min alpha-thrombin exposure and decayed to basal levels within 90 min. In contrast, ICAM-1 activity increased at 30 min and remained elevated for 120 min after alpha-thrombin challenge. The initial endothelial adhesivity was dependent on P-selectin expression since PMN adhesion occurring within the first 30 min after alpha-thrombin challenge was inhibited by mAb G1. The later prolonged PMN adhesion was ICAM-1 dependent since this response was inhibited by mAb RR1/1 and to the same degree by the anti-CD18 mAb IB4. Anti-ELAM-1 mAb BB11 had no effect on adhesion of PMN to the alpha-thrombin-challenged cells. The initial P-selectin expression and PMN adhesion responses were reproduced by the 14-amino peptide (SFLLRNPNDKYEPF) (thrombin-receptor activity peptide; TRP-14) which comprised the NH2 terminus created by thrombin's proteolytic action on its receptors. However, TRP-14-induced PMN adhesion was transient, and TRP-14 did not cause ICAM-1 expression. The ICAM-1-dependent PMN adhesion mediated by alpha-thrombin was protein synthesis independent since ICAM-1 expression and PMN adhesion were not inhibited by cycloheximide pretreatment of HUVEC. Moreover, Northern blot analysis indicated absence of ICAM-1 mRNA signal up to 180 min after alpha-thrombin challenge. In conclusion, thrombin-induced endothelial adhesivity involves early- and late-phase responses. The initial reversible PMN adhesion is mediated by rapid P-selectin expression via TRP-14 generation. Thrombin-induced PMN adhesion is stabilized by a protein synthesis-independent upregulation of the constitutive ICAM-1 activity which enables the interaction of ICAM-1 with the CD18 beta 2 integrin on PMN.
The purpose of this study was the development of a model of embolic stroke with high reproducibility concerning infarct volume. In 37 male Sprague-Dawley rats, the internal carotid artery was embolized with in vitro preformed suspensions of autologous microemboli resembling arterial thrombi. With a method of continuous flow through the carotid arterial catheter, reflux of blood with uncontrolled clotting and embolization was avoided, thereby providing control animals free of ischemic damage. The embolized animals had arterial occlusions on angiograms immediately after embolization and no spontaneous recanalization on angiograms 2 h later. The cerebral blood flow measured by the intra-arterial 133Xe injection method decreased to 21-37% of baseline values. All embolized animals developed hemiparesis with spontaneous circling behavior, embolization with more than 150 microliters clot suspension resulted in hemispherical infarcts. There was a strong statistically significant correlation between amount of emboli, rate of vascular occlusion, and volume of infarcted tissue. This is the first model presented utilizing autologous in vitro microemboli imitating "white" arterial thrombi. The animals developed infarction, resembling human stroke.
Thrombolytic agents may be useful in the treatment of cerebral ischemia caused by arterial thrombosis or embolic occlusion. A trial of intravenous human tissue-type plasminogen activator (rt-PA) was carried out in seven male Sprague-Dawley rats subjected to embolic cerebral ischemia, with eight control animals. One-hour-old autogenous blood clot was injected into the internal carotid artery. A 30-minute infusion of 10 micrograms/kg/minute of rt-PA or saline followed. Areas of ischemia at two hours post-embolization were assessed by digital image processing of serial iodo-14C-antipyrine autoradiographic images. The volumes of "no-flow" (NF) and "low-flow" (LF) regions were calculated. One animal in each group suffered no detectable ischemia; the remainder had well-defined regions of middle and posterior cerebral artery ischemia. No animal sustained a hemorrhagic lesion. Treatment produced no noticeable effect on the patency of cervical vessels. Total NF and LF volumes were less for the treated group but did not reach statistical significance by t-test. In middle cerebral distribution sections, however, LF volume was significantly less (p less than 0.05) for treated animals (150 vs. 191 mm3), primarily due to a more significant decrease in LF volume in the anterior-middle cerebral overlap zone (47 vs. 90 mm3; p less than 0.025). Fibrinogen levels were not altered by drug treatment (p greater than 0.30).
Fibrinogen degradation, fibrin polymerisation, and the insertion of cross links into fibrin by fibrin stabilising factor lead to the appearance of new antigenic determinants. Antibodies against these antigenic sites may react specifically with the derivatives but not with the parent molecules. We have utilised a monoclonal antibody, which interacts with the cross linked fragment D dimer and related high molecular weight fibrin derivatives, to develop an enzyme immunoassay which measures cross linked fibrin derivatives in plasma and serum using D dimer as standard. Mean concentration in plasma from normal subjects was 75 ng/ml with an upper limit of about 144 ng/ml. Concentrations in patients with pulmonary embolism, deep venous thrombosis, arterial thromboembolism, and disseminated intravascular coagulation were raised in all cases. Confirmation of the specific increase of cross linked fibrin derivatives in patients with disseminated intravascular coagulation was obtained by parallel monitoring of their fibrin degradation products in serum using affinity chromatography and sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. In many patients the plasma concentrations greatly exceeded the serum values of cross linked fibrin degradation products, suggesting that the procedure can measure fibrin derivatives in plasma which are absent from serum.
A simple venous thrombosis model in rabbits was used for the quantitative evaluation of the thrombolytic effect of human extrinsic (tissue-type) plasminogen activator as compared with urokinase.A thrombus was formed in an isolated segment of the jugular vein from a mixture of (125)I-labeled fibrinogen, whole rabbit blood, and thrombin. In order to immobilize the thrombus during lysis, it was formed around a woolen thread introduced longitudinally in the lumen of the vein. Thrombotic extension of the clot was prevented by subcutaneous injection of heparin. The extent of thrombolysis was measured as the difference between the radioactivity introduced in the clot and that recovered in the vein segment at the end of the experiment. In control animals the extent of thrombolysis was 5.6+/-1.4% (n = 5) after 6 h, 14.5+/-1.7% (n = 10) after 30 h, 16.0+/-1.5% (n = 11) after 78 h, and 48.1+/-2.7% (n = 10) after 174 h (mean+/-SEM). Extrinsic (tissue-type) plasminogen activator, highly purified from the culture fluid of a human melanoma cell line, was administered systemically or locally over a time period of 4 h and the percent thrombolysis measured 2 h after the end of the infusion. One- and two-chain extrinsic plasminogen activator had very similar thrombolytic potency. Systemic infusion resulted in a dose-dependent degree of thrombolysis. The activator-induced thrombolysis, after infusion of 100,000 IU ( congruent with1 mg protein), was approximately 75% for fresh clots, 35% for 1-d-old clots, 30% for 3-d-old clots, and 50% for 7-d-old clots. The thrombolytic activity of urokinase was more than five times lower than that of extrinsic plasminogen activator: Infusion of 500,000 IU resulted in approximately 40% lysis of fresh clots and 25% of 1-3-d-old clots, while 7-d-old clots appeared to have become resistent to urokinase. Local infusion resulted in a 5-10 times higher thrombolytic effect of both extrinsic plasminogen activator and urokinase. Thrombolysis with extrinsic plasminogen activator was not associated with systemic activation of the fibrinolytic system as evidenced by unaltered plasma levels of fibrinogen, plasminogen, and alpha(2)-antiplasmin. Systemic infusion of urokinase resulted in significant thrombolysis only at doses that were associated with disseminated plasminogen activation. Local infusion of urokinase required a 5-10-fold higher dose than extrinsic plasminogen activator to obtain a similar degree of thrombolysis, which also occurred in the absence of systemic activation of the fibrinolytic system. It is concluded that the extent of thrombolysis by extrinsic plasminogen activator is mainly determined by the dose of activator and its delivery in the vicinity of the thrombus and much less by the age of the thrombus or the molecular form of the activator. Extrinsic plasminogen activator appears to be superior to urokinase because of its higher (5-10-fold) specific thrombolytic activity and the absence of systemic activation of the fibrinolytic system, which results in defibrinogenation and a bleeding tendency.
PADGEM protein (PADGEM), also known as GMP140, is a platelet alpha- granule membrane protein that is translocated to the external membrane after platelet activation. Although the biosynthesis of this protein was originally thought to be confined to megakaryocytes, the synthesis of PADGEM in endothelial cells was recently demonstrated (McEver et al: Blood 70:1974a, 1987). We now describe the subcellular localization of this protein in endothelial cells. Immunofluorescence staining of permeabilized human umbilical vein endothelial cells with KC4, a well characterized monoclonal antibody to PADGEM, showed positively stained elongated structures similar in distribution and shape to Weibel-Palade bodies. Their identity as Weibel-Palade bodies was confirmed by double label immunofluorescence using KC4 and a polyclonal antiserum to von Willebrand factor (vWf), a protein known to be specifically stored in these organelles. All Weibel-Palade bodies were found to contain PADGEM. In contrast to strong perinuclear staining produced with anti- vWf antibodies, no significant perinuclear staining was obtained with KC4, indicating that relatively little PADGEM is present in the endoplasmic reticulum and in the Golgi apparatus. In endothelial cells treated with secretagogues that stimulate vWf release the elongated structures positive for PADGEM disappeared, further identifying these structures as Weibel-Palade bodies. This observation extends the parallels between Weibel-Palade bodies and alpha-granules and suggests a possible functional association between vWf and PADGEM.
The role of defective fibrinolysis caused by elevated activity of plasminogen activator inhibitor-1 (PAI-1) in promoting fibrin deposition in vivo has not been well established. The present study compared the efficacy of thrombin or ancrod, a venom-derived enzyme that clots fibrinogen, to induce fibrin formation in rabbits with elevated PAI-1 levels. One set of male New Zealand rabbits received intravenous endotoxin to increase endogenous PAI-1 activity followed by a 1-hour infusion of ancrod or thrombin; another set of normal rabbits received intravenous human recombinant PAI-1 (rPAI-1) during an infusion of ancrod or thrombin. Thirty minutes after the end of the infusion, renal fibrin deposition was assessed by histopathology. Animals receiving endotoxin, rPAI-1, ancrod, or thrombin alone did not develop renal thrombi. All endotoxin-treated rabbits developed fibrin deposition when infused with ancrod (n = 4) or thrombin (n = 6). Fibrin deposition occurred in 7 of 7 rabbits receiving both rPAI-1 and ancrod and in only 1 of 6 receiving rPAI-1 and thrombin (P “ .01). In vitro, thrombin but not ancrod was inactivated by normal rabbit plasma and by purified antithrombin III or thrombomodulin. The data indicate that elevated levels of PAI-1 promote fibrin deposition in rabbits infused with ancrod but not with thrombin. In endotoxin-treated rabbits, fibrin deposition that occurs with thrombin infusion may be caused by decreased inhibition of procoagulant activity and not increased PAI-1 activity.
We investigated the effect of an anti-CD11b monoclonal antibody (IB6c) on ischemic cell damage after transient (2h) and permanent middle cerebral artery (MCA) occlusion in the rat. Groups studied were: permanent MCA occlusion treated with IB6c (n=8) and with isotype matched control antibody (n=8) and sacrificed 48 hours after onset of ischemia; transient MCA occlusion: animals treated with IB6c or isotype control antibody were sacrificed at 48 hours (n=16) or at 1 week (n=16) after initiation of ischemia. Brains were removed for measurement of the ischemic lesion. A significant reduction in volume (~37%) of ischemic lesion was found in all animals treated with IB6c compared to the vehicle control groups (p<0.05). In contrast, in permanent ischemia, the lesion was not altered by IB6c administration. Our data indicate that treatment with an anti CD11b/18 monoclonal antibody significantly reduces ischemic cell damage after 2h of transient ischemia but not after permanent ischemia.
Thrombin-induced expression of endothelial adhesivity toward neutrophils (PMN) was studied using human umbilical vein endothelial cells (HUVEC). HUVEC were challenged with human alpha-thrombin for varying durations up to 120 min, after which the cells were fixed with 1% paraformaldehyde and 51Cr-labeled human PMN were added to determine PMN adhesion. Endothelial adhesivity increased within 15 min after alpha-thrombin exposure, and the response persisted up to 120 min. Expression of endothelial adhesion proteins, P-selectin (GMP-140, PADGEM, CD62), and intercellular adhesion molecule-1 (ICAM-1; CD54) on the endothelial surface was quantitated by increase in the specific binding of anti-P-selectin mAb G1 and anti-ICAM-1 mAb RR1/1 labeled with 125I. P-selectin expression was maximal at 5-15 min alpha-thrombin exposure and decayed to basal levels within 90 min. In contrast, ICAM-1 activity increased at 30 min and remained elevated for 120 min after alpha-thrombin challenge. The initial endothelial adhesivity was dependent on P-selectin expression since PMN adhesion occurring within the first 30 min after alpha-thrombin challenge was inhibited by mAb G1. The later prolonged PMN adhesion was ICAM-1 dependent since this response was inhibited by mAb RR1/1 and to the same degree by the anti-CD18 mAb IB4. Anti-ELAM-1 mAb BB11 had no effect on adhesion of PMN to the alpha-thrombin-challenged cells. The initial P-selectin expression and PMN adhesion responses were reproduced by the 14-amino peptide (SFLLRNPNDKYEPF) (thrombin-receptor activity peptide; TRP-14) which comprised the NH2 terminus created by thrombin's proteolytic action on its receptors. However, TRP-14-induced PMN adhesion was transient, and TRP-14 did not cause ICAM-1 expression. The ICAM-1-dependent PMN adhesion mediated by alpha-thrombin was protein synthesis independent since ICAM-1 expression and PMN adhesion were not inhibited by cycloheximide pretreatment of HUVEC. Moreover, Northern blot analysis indicated absence of ICAM-1 mRNA signal up to 180 min after alpha-thrombin challenge. In conclusion, thrombin-induced endothelial adhesivity involves early- and late-phase responses. The initial reversible PMN adhesion is mediated by rapid P-selectin expression via TRP-14 generation. Thrombin-induced PMN adhesion is stabilized by a protein synthesis-independent upregulation of the constitutive ICAM-1 activity which enables the interaction of ICAM-1 with the CD18 beta 2 integrin on PMN.
Thrombolytic therapy for acute ischemic stroke has been approached cautiously because there were high rates of intracerebral hemorrhage in early clinical trials. We performed a randomized, double-blind trial of intravenous recombinant tissue plasminogen activator (t-PA) for ischemic stroke after recent pilot studies suggested that t-PA was beneficial when treatment was begun within three hours of the onset of stroke.
METHODS:
The trial had two parts. Part 1 (in which 291 patients were enrolled) tested whether t-PA had clinical activity, as indicated by an improvement of 4 points over base-line values in the score of the National Institutes of Health stroke scale (NIHSS) or the resolution of the neurologic deficit within 24 hours of the onset of stroke. Part 2 (in which 333 patients were enrolled) used a global test statistic to assess clinical outcome at three months, according to scores on the Barthel index, modified Rankin scale, Glasgow outcome scale, and NIHSS:
RESULTS:
In part 1, there was no significant difference between the group given t-PA and that given placebo in the percentages of patients with neurologic improvement at 24 hours, although a benefit was observed for the t-PA group at three months for all four outcome measures. In part 2, the long-term clinical benefit of t-PA predicted by the results of part 1 was confirmed (global odds ratio for a favorable outcome, 1.7; 95 percent confidence interval, 1.2 to 2.6). As compared with patients given placebo, patients treated with t-PA were at least 30 percent more likely to have minimal or no disability at three months on the assessment scales. Symptomatic intracerebral hemorrhage within 36 hours after the onset of stroke occurred in 6.4 percent of patients given t-PA but only 0.6 percent of patients given placebo (P < 0.001). Mortality at three months was 17 percent in the t-PA group and 21 percent in the placebo group (P = 0.30).
CONCLUSIONS:
Despite an increased incidence of symptomatic intracerebral hemorrhage, treatment with intravenous t-PA within three hours of the onset of ischemic stroke improved clinical outcome at three months.
• Investigators have tried to limit ischemic cerebral infarct size by pharmacologic and surgical means with mixed results. Thrombolytic (fibrinolytic) therapy has been used in the past with unfavorable outcome. With advances in clinical and radiologic assessment and new knowledge of the pathophysiology of brain ischemia, thrombolytic therapy has now become a feasible pharmacologic intervention in acute stroke. Central nervous system hemorrhage, the most dread complication of fibrinolytic therapy, is rare in patients with acute myocardial infarction favorably treated with these agents. Risk of hemorrhagic transformation of ischemic cerebral infarcts is related to size, location, and age of patient. Anticoagulation therapy may increase its size, but not its likelihood. The development of clot-specific agents, such as tissue-type plasminogen activator, and careful patient selection make fibrinolytic therapy safe and potentially effective in acute stroke.
• Tissue plasminogen activator (tPA) has become available for pharmacologic use, and it appears to produce relatively fewer hemorrhagic complications than the previously available, less specific thrombolytic agents. We tested the effects of tPA in several models of embolic stroke and found that neurologic damage was reduced when the drug was administered as late as 45 minutes after cerebral embolic occlusion. The mechanism of therapeutic efficacy of tPA was probably thrombolysis. Drug-induced hemorrhages did not occur when therapy was started within four hours after the onset of vascular occlusion. These results suggest that tPA may be useful for thrombolytic therapy of embolic stroke if the drug is administered rapidly after the onset of vascular occlusion.
FLASH (Fast Low-Angle SHot) imaging is a new method for rapid NMR imaging which has been demonstrated to provide abdominal images without artifacts due to respiratory or peristaltic motions. The sequence typically employs 15° radiofrequency excitation pulses and acquires a free induction decay signal in the form of a gradient echo. Here FLASH images are recorded in the presence of dynamic processes with time constants even smaller than the measuring time of about 2 s for an image with a 128 × 128-pixel resolution. Experiments are carried out on flow phantoms and on rabbits yielding heart images without gating of the cardiac motion. © 1986 Academic Press, Inc.
Intravenous administration of tissue plasminogen activator immediately after the injection of numerous small blood clots into
the carotid circulation in rabbit embolic stroke model animals caused a significant reduction in neurological damage. In vitro
studies indicate that tissue plasminogen activator produced substantial lysis of clots at concentrations comparable to those
expected in vivo, suggesting that this may be the mechanism of action of this drug. Drug-induced hemorrhages were not demonstrable.
Tissue plasminogen activator may be of value for the immediate treatment of embolic stroke.
Data from 694 patients hospitalized with stroke were entered in a prospective, computer-based registry. Three hundred and sixty-four patients (53 percent) were diagnosed as having thrombosis, 215 (31 percent)as having cerebral embolism 70 (10 percent) as having intracerebral hematoma, and 45 (6 percent) as having subarachnoid hemorrhage from aneurysm or arteriovenous malformations. The 364 patients diagnosed as having thrombosis were divided into 233 (34 percent of all 694 patients) whose thrombosis was thought to involve a large artery and 131 (19 percent) with lacunar infarction. Many of the findings in this study were comparable to those in previous registries based on postmortem data. New observations include the high incidence of lacunes and cerebral emboli, the absence of an identifiable cardiac origin in 37 percent of all emboli, a nonsudden onset in 21 percent of emboli, and the occurrence of vomiting at onset in 51 percent and the absence of headache at onset in 67 percent of hematomas.
Fifteen patients admitted to Philadelphia General Hospital with acute strokes had repeated measurements of cerebral blood flow measured by the 133Xenon inhalation method. A progressive decline in cerebral blood flow in both hemispheres was observed during the first week after infarction in twelve of these patients. This decline could be partially explained by loss of autoregulation, but could not be correlated with level of consciousness, clinical status of PCO2. This progressive decline in flow in the non-ischemic hemisphere indicates a process more complex than a simple destruction of axonal afferants to neurons as implied by the term diaschisis. The flow changes in the non-ischemic hemisphere are likely caused by a combination of the immediate effects of decreased neuronal stimulation modified by loss of autoregulation, release of vasoactive substances, cerebral edema, and other factors.
A pathological examination was performed on normotensive rats (NTR) and spontaneously hypertensive rats (SHR) following bilateral common carotid artery ligation. After ligation, diffuse and extensive cerebral infarcts in the carotid artery territory occurred frequently in SHR, while NTR occasionally had well-circumscribed small infarcts. The posterior communicating arteries, which are the major anastomotic channels connecting the carotid and vertebrobasilar systems, did not show any anomalies and were well developed in SHR and NTR. Vascular changes secondary to hypertension, such as fibrinoid necrosis or thickening of the wall, were not observed in SHR. Because of the paucity of structural difference of the blood vessels, the more diffuse and extensive cerebral infarcts in SHR after carotid occlusion were attributed to the hemodynamic difference rather than the morphological difference between the two groups. The results of the present experiment suggest that hypertension per se, i.e., hemodynamic factors, may be operative for the development of cerebral infarction.
A technique has been developed for proton magnetic resonance imaging (MRI) of perfusion, using water as a freely diffusable tracer, and its application to the measurement of cerebral blood flow (CBF) in the rat is demonstrated. The method involves labeling the inflowing water proton spins in the arterial blood by inverting them continuously at the neck region and observing the effects of inversion on the intensity of brain MRI. Solution to the Bloch equations, modified to include the effects of flow, allows regional perfusion rates to be measured from an image with spin inversion, a control image, and a T1 image. Continuous spin inversion labeling the arterial blood water was accomplished, using principles of adiabatic fast passage by applying continuous-wave radiofrequency power in the presence of a magnetic field gradient in the direction of arterial flow. In the detection slice used to measure perfusion, whole brain CBF averaged 1.39 +/- 0.19 ml.g-1.min-1 (mean +/- SEM, n = 5). The technique's sensitivity to changes in CBF was measured by using graded hypercarbia, a condition that is known to increase brain perfusion. CBF vs. pCO2 data yield a best-fit straight line described by CBF (ml.g-1.min-1) = 0.052pCO2 (mm Hg) - 0.173, in excellent agreement with values in the literature. Finally, perfusion images of a freeze-injured rat brain have been obtained, demonstrating the technique's ability to detect regional abnormalities in perfusion.
We investigated plasma levels of D-dimer products of crosslinked fibrin degradation products, thrombin-antithrombin III complex, and plasmin-alpha 2-antiplasmin complex for detecting coagulation system activation in ischemic stroke patients to determine the possible effect of age on these marker levels.
We measured plasma levels of these three markers in 54 acute ischemic stroke patients within 5 days of stroke onset, in 44 chronic ischemic stroke patients over 3 months from onset, and in 50 age-matched healthy subjects. We divided the stroke patients into two subgroups, those with visible occlusion and those with nonvisible occlusion having obstruction of the major cerebral artery.
The plasma levels of these three markers were significantly (p less than 0.01) higher in the stroke patients than in controls. Significant differences did not exist at any level between the patients and controls in the younger-aged subjects (less than or equal to 64 years of age), but did exist in the older-aged subjects (greater than or equal to 75 years of age). An age-related increase of the marker levels was noted between stroke patients and controls. No significant difference in the three markers was found among any of the stroke patients.
Increased levels of these markers in stroke patients seem to be related mostly to age.
There has been major interest in the potential interaction between blood coagulation and inflammation. Most of the effort has focused on cellular interactions involving platelets and polymorphonuclear leukocytes (PMNS). The recent discovery of tissue kallikrein(TK) activity in PMNs prompted the study of the possible role of thrombin(IIa) in this process. Human PMNs were isolated by density gradient centrifugation. Human IIa was compared with fMLP with respect to chemotaxis and enzyme release. Results from the challenges by IIa and fMLP were compared to a NaCl control using Student's paired t-test. IIa was a potent chemotactic agent for PMNs (p less than or equal to 0.0121) and stimulated the release of TK (p less than or equal to 0.0001) as determined by hydrolysis of S-2266. FMLP significantly stimulated PMN chemotaxis (p less than or equal to 0.0028) but had no effect on TK release. Release of TK was confirmed by Western Blot analysis and 35S-methionine incorporation into a 35 KD protein after IIa challenge. These results demonstrate that IIa is chemotactic for PMNs and can cause release of tissue kallikrein demonstrating a direct role for blood coagulation in the regulation of the inflammatory response.
We assayed plasma concentrations of fibrinogen, fibrinopeptide A, plasmin-alpha 2 plasmin inhibitor complex, D dimer, and antithrombin III activity in 40 patients with cerebral thrombosis and nine patients with cerebral embolism during the acute (less than 7 days), subacute (7-27 days), and chronic (greater than or equal to 28 days) periods and compared these with 69 controls. In cerebral thrombosis, fibrinogen and fibrinopeptide A levels were elevated significantly in all stages (p less than 0.001), whereas plasmin-alpha 2 plasmin inhibitor complex and D dimer levels were elevated significantly in the subacute and chronic periods. The antithrombin III activity was significantly decreased in the acute stage. The elevation of fibrinogen and plasmin-alpha 2 plasmin inhibitor complex levels in the acute stage was significantly greater in patients with an infarct size greater than 10 mm2 compared to patients with an infarct size less than 10 mm2. We observed similar changes in patients with cerebral embolism. These results suggest that enhanced coagulation exists at all stages and endogenous fibrinolysis is activated in the subacute and chronic periods in a large proportion of patients with cerebral thrombosis and embolism.
To develop a simple, relatively noninvasive small-animal model of reversible regional cerebral ischemia, we tested various methods of inducing infarction in the territory of the right middle cerebral artery (MCA) by extracranial vascular occlusion in rats. In preliminary studies, 60 rats were anesthetized with ketamine and different combinations of vessels were occluded; blood pressure and arterial blood gases were monitored. Neurologic deficit, mortality rate, gross pathology, and in some instances, electroencephalogram and histochemical staining results were evaluated in all surviving rats. The principal procedure consisted of introducing a 4-0 nylon intraluminal suture into the cervical internal carotid artery (ICA) and advancing it intracranially to block blood flow into the MCA; collateral blood flow was reduced by interrupting all branches of the external carotid artery (ECA) and all extracranial branches of the ICA. In some groups of rats, bilateral vertebral or contralateral carotid artery occlusion was also performed. India ink perfusion studies in 20 rats documented blockage of MCA blood flow in 14 rats subjected to permanent occlusion and the restoration of blood flow to the MCA territory in six rats after withdrawal of the suture from the ICA. The best method of MCA occlusion was then selected for further confirmatory studies, including histologic examination, in five additional groups of rats anesthetized with halothane. Seven of eight rats that underwent permanent occlusion of the MCA had resolving moderately severe neurologic deficits (Grade 2 of 4) and unilateral infarcts averaging 37.6 +/- 5.5% of the coronal sectional area at 72 hours after the onset of occlusion.(ABSTRACT TRUNCATED AT 250 WORDS)
To assess the time course of thrombosis and fibrinolysis after acute stroke, we measured concentrations of fibrinopeptide A (FpA), B-beta 1-42 peptide (B-beta 1-42), B-beta 15-42 peptide (B-beta 15-42), and crosslinked D-dimer (XDP) in 31 patients at varying times following acute ischemic stroke and in 13 neurologically stable patients with chronic strokes. FpA levels were markedly elevated during the first week after stroke and declined slowly during the first month. Mean FpA levels were not significantly elevated in chronic stroke patients. Mean XDP levels were slightly elevated during the first week and increased during the next 2 weeks after stroke. B-beta 1-42 and B-beta 15-42 levels were not elevated at any time following acute stroke. Our data suggest that fibrin formation greatly exceeds endogenous fibrinolysis during the acute phase of ischemic stroke. Endogenous fibrinolysis develops slowly following stroke. Prolonged elevation of FpA concentration suggests that thrombin activity and fibrin formation continue for up to 4 weeks in some patients with ischemic stroke.
We describe a new focal ischemia model consisting of unilateral middle cerebral artery occlusion with a silicone rubber cylinder attached to a nylon surgical thread inserted through the internal carotid artery in rats. Recirculation was accomplished by pulling the thread out of the artery. We evaluated the reliability of this model and studied the influence of reperfusion of the brain by measuring regional cerebral blood flow in 30 rats and by using conventional neuropathologic methods after different periods of occlusion in 48 rats. The anterior neocortex and the lateral part of the caudate putamen, which were supplied by the occluded middle cerebral artery, were the regions most frequently damaged. After 1 hour of occlusion in five rats, in the cortex supplied by the occluded artery mean +/- SD blood flow was 0.19 +/- 0.08 ml/g/min (approximately 15% of that in the corresponding region of five sham-operated control rats), and mild scattered ischemic cell change was observed. Three (n = 5) or six (n = 5) hours of occlusion reduced blood flow more severely and caused severe ischemic cell changes in the cortex supplied by the occluded artery in proportion to the duration of ischemia. Characteristically, in five rats subjected to 3 hours of occlusion followed by 3 hours of recirculation, blood flow was restored and spongy edematous change was observed in the cortex supplied by the recirculated artery. This change resulted in hypoperfusion of the neighboring cortical region surrounding the recirculated area. Our model should be useful in various investigations of the influence of reperfusion on focal ischemic brain injury.
We developed a new animal model of stroke which resembles human stroke more closely than existing models. We described the pathology produced in the brain following platelet embolism, previously described only in the retina. The common carotid artery of the rat was irradiated for 6.5 minutes with an argon laser at 514.5 nm after intravenous injection of a photosensitizing agent, rose bengal. A retinal embolus was seen in 1 rat 5 minutes after irradiation. A nonocclusive platelet thrombus was present in the carotid artery 50 minutes after irradiation, with almost all the platelet thrombus being cleared 24 hours later. Acute (1 to 10 days) changes in the brain included 44 small infarcts in 12/13 rats, cortical arterioles occluded with platelets and thickening of small vessels in normotensive rats. Chronic (4 to 12 weeks) changes included lacunes in the brains of normotensive rats and intimal proliferation of smooth muscle in the carotid artery. This is the 1st animal model of (1) stroke with emboli produced in vivo rather than injected into the carotid, (2) intimal proliferation of smooth muscle without invasion of the vessel, and (3) lacunes. This model provides results important to the laboratory study of stroke.
Systemic fibrinolytic therapy for acute stroke is no longer recommended because of resulting systemic fibrinolysis and the risk of intracerebral hemorrhage. Human tissue-type plasminogen activator (TPA) is a native enzyme that converts plasminogen to plasmin with subsequent clot lysis. The affinity for plasminogen is increased several-fold when the substrate is bound to fibrin. At appropriate dosage, “clot-specific” throm-bolysis may be achieved at the surface of the thrombus without creating systemic fibrinolysis.
The authors designed a study to evaluate the effect of intravenous TPA administered 2 hours after acute thromboembolic stroke in rats. This time course was chosen to simulate an analogous clinical situation. Middle cerebral artery embolic stroke was caused by intracarotid injection of 0.025 cc of human blood clot in 16 rats. Regional cerebral blood flow, measured by the hydfogen clearance technique, and electroencephalographic (EEG) recordings were obtained every 30 minutes for 5 hours after thromboembolism. Eight rats received a 1-hour infusion of intravenous TPA (1.5 mg/kg) 2 hours after injection of emboli. Ipsilateral blood flow increased significantly within 30 minutes after intravenous TPA and reached preembolic levels within 60 minutes. Blood flow did not improve in the eight control rats throughout the experiment. Power spectral analysis of the EEG recordings showed improvement in the treated group compared to the control group. Postmortem angiography revealed proximal middle cerebral artery occlusion in control animals and patent middle cerebral arteries in TPA-treated animals. Serum fibrinogen and fibrin split products were unchanged in both groups, indicating the absence of systemic fibrinolysis. There were no intracerebral hemorrhages.
It is concluded that, in this rat model, TPA increases blood flow with subsequent improvement in the EEG recording after thromboembolic stroke without evidence of systemic fibrinolysis. Intravenous TPA may be useful in the treatment of acute stroke in man.
Recent success with thrombolytic therapy for acute myocardial infarction has stimulated interest in its use for stroke. To determine the hemorrhagic potential of thrombolytic therapy in experimental cerebral infarction, we compared a group of tissue plasminogen activator-treated rabbits (n = 4) with 2 groups of streptokinase-treated rabbits (n = 6 in each), as well as with 3 groups of heparin-treated rabbits (n = 5 in each) and untreated controls (n = 12). Focal cerebral infarction was produced in rabbits by occlusion of the right common carotid and middle cerebral arteries coupled with 2 hours of halothane-induced hypotension. Treatment with heparin or thrombolytic agents began 24 hours after occlusion. One additional group was treated with streptokinase 1 hour after occlusion (n = 6) to determine the hemorrhagic potential of thrombolytic agents in evolving infarction. Rabbits were killed 29-33 hours after occlusion, and brain sections were examined using light microscopy. The results demonstrate that microscopic hemorrhage is frequently present in infarcted tissue irrespective of treatment. Gross cerebral hemorrhage did not occur in untreated rabbits or in rabbits treated with streptokinase 1 hour after occlusion. Only rabbits treated with streptokinase, tissue plasminogen activator, or excessive doses of heparin 24 hours after occlusion, at a time when cerebral infarction was well established, exhibited gross hemorrhage in the area of infarction. These data suggest that treatment of ischemic stroke with thrombolytic agents carries an increased risk of cerebral hemorrhage unless the agents are given early after the onset of symptoms.
Tissue plasminogen activator (tPA) has become available for pharmacologic use, and it appears to produce relatively fewer hemorrhagic complications than the previously available, less specific thrombolytic agents. We tested the effects of tPA in several models of embolic stroke and found that neurologic damage was reduced when the drug was administered as late as 45 minutes after cerebral embolic occlusion. The mechanism of therapeutic efficacy of tPA was probably thrombolysis. Drug-induced hemorrhages did not occur when therapy was started within four hours after the onset of vascular occlusion. These results suggest that tPA may be useful for thrombolytic therapy of embolic stroke if the drug is administered rapidly after the onset of vascular occlusion.
Thrombolysis with tissue plasminogen activator (tPA) has been used to treat myocardial infarction and pulmonary embolism in humans. This plasminogen activator may also be useful in treating certain strokes. We infused tPA or saline in rabbits 15 minutes after selective internal carotid artery embolization with 18-hour aged autologous clot. By serial angiography, the tPA-treated group demonstrated rapid angiographic reperfusion of the occluded vascular territory in 7 of 8 animals, whereas none of 6 saline controls did. None of the animals in either group developed macroscopic cerebral hemorrhage. Both groups showed cerebral infarction, predominantly in the territory of the occluded vessel; the extent of infarction did not differ between tPA-treated animals and controls. Early tPA therapy can allow reperfusion of occluded cerebral arteries safely and effectively in a rabbit cerebral embolization model.
Investigators have tried to limit ischemic cerebral infarct size by pharmacologic and surgical means with mixed results. Thrombolytic (fibrinolytic) therapy has been used in the past with unfavorable outcome. With advances in clinical and radiologic assessment and new knowledge of the pathophysiology of brain ischemia, thrombolytic therapy has now become a feasible pharmacologic intervention in acute stroke. Central nervous system hemorrhage, the most dreaded complication of fibrinolytic therapy, is rare in patients with acute myocardial infarction favorably treated with these agents. Risk of hemorrhagic transformation of ischemic cerebral infarcts is related to size, location, and age of patient. Anticoagulation therapy may increase its size, but not its likelihood. The development of clot-specific agents, such as tissue-type plasminogen activator, and careful patient selection make fibrinolytic therapy safe and potentially effective in acute stroke.
In 65 cases of ischemic cerebral infarction, CT scans and quantitative assessments of the neurological disturbances were undertaken at specific intervals during the 4 week period after stroke. Forty-three patients underwent lumbar puncture to determine the serum/CSF albumin ratio. The etiology of the infarction was evaluated on the basis of angiographic, ultrasonic and cardiologic findings. A hemorrhagic transformation of the infarction occurred in 28 patients, eleven within the first week, and 15 within the second. Risks of hemorrhage were correlated with a severe neurological deficit, disturbance of consciousness, large infarction with a mass effect, enhancement of contrast medium in CT (especially if occurring early), involvement of the cortex, and distinct blood/CSF barrier disturbances. Cardiac embolism was a frequent etiology in patients with secondary hemorrhagic infarction, especially when transformation occurred within the first week after stroke. In addition to a heterogeneous pattern of hemorrhage, frank hematoma predominated in those infarcts which underwent early transformation, while those transforming late often showed less hyperdense cortical hemorrhagic changes. Deterioration evident on clinical evaluation was caused by the hemorrhagic transformation in three cases, in each instance within the first week after stroke.
Embolic stroke was induced in rabbits using autologous blood clot. One hour after stroke, animals received heparin anticoagulation (AC) for five hours (acute AC) or five days (chronic AC). Animals received excessive AC (partial thromboplastin time greater than 3.0 times control), adequate AC (partial thromboplastin time, 1.2 to 2.5 times control), or saline. After the animals were killed, the brains were examined for macroscopic evidence of intracerebral hemorrhage. There was no significant increase over control in the incidence or severity of hemorrhage in any of the four treatment groups. The data suggest that heparin AC does not promote intracerebral hemorrhage after experimental embolic stroke.
FLASH (Fast Low-Angle SHot) imaging is a new method for rapid NMR imaging which has been demonstrated to provide abdominal images without artifacts due to respiratory or peristaltic motions. The sequence typically employs 15 degrees radiofrequency excitation pulses and acquires a free induction decay signal in the form of a gradient echo. Here FLASH images are recorded in the presence of dynamic processes with time constants even smaller than the measuring time of about 2 s for an image with a 128 X 128-pixel resolution. Experiments are carried out on flow phantoms and on rabbits yielding heart images without gating of the cardiac motion.
In 48 patients dying within 15 days following a supra-tentorial cerebral infarct, the presence of hemorrhagic infarction at autopsy was related to a cardiac embolic cause of the infarct, and to the cause of death. Hemorrhagic infarcts were more common among patients dying from brain herniation than among those dying from a non-cerebral cause. Cardiac embolic strokes were more often hemorrhagic at autopsy than strokes without such cause; this could be explained by a significant higher rate of brain herniation and death after embolic stroke. On the other hand infarcts with extended hemorrhages more often tended to have a cardiac than a non-cardiac cause. These data, together with earlier clinical findings suggest that autopsy studies are biased in relating hemorrhagic infarction almost exclusively to a cardiac embolic cause of stroke, although cardiac emboli may produce more extended hemorrhages.
To study the effects of antithrombotic therapy in experimental stroke, we have characterized a baboon model of acute cerebrovascular thrombosis. In this model an inflatable silastic balloon cuff has been implanted by transorbital approach around the right middle cerebral artery (MCA), proximal to the take-off of the lenticulostriate arteries (LSA). Inflation of the balloon for 3 hours in six animals produced a stereotypic sustained stroke syndrome characterized by contralateral hemiparesis. An infarction volume of 3.2 +/- 1.5 cm3 in the ipsilateral corpus striatum was documented by computerized tomographic (CT) scanning at 10 days following stroke induction and 3.9 +/- 1.9 cm3 (n = 4) at 14 days by morphometric neuropathologic determinations of brain specimens fixed in situ by pressure-perfusion with 10% buffered formalin. Immediate pressure-perfusion fixation following deflation of the balloon was performed in 16 additional animals given Evans blue dye intravenously prior to the 3 hour MCA balloon occlusion. Light microscopy and transmission electron microscopy consistently confirmed the presence of thrombotic material occluding microcirculatory branches of the right LSA in the region of Evans blue stain, but not those of the contralateral corpus striatum. When autologous 111In-platelets were infused intravenously in four animals from the above group prior to the transient 3 hour occlusion of the right MCA, gamma scintillation camera imaging of each perfused-fixed whole brain demonstrated the presence of a single residual focus of 111In-platelet activity involving only the Evans blue-stained right corpus striatum. Focal right hemispheric activity was equivalent to 0.55 +/- 0.49 ml of whole blood, and the occlusion score derived from histologic examination of the microcirculation of the Evans blue-stained corpus striatum averaged 34.8 +/- 2.8. Similar 111In-platelet imaging and histologic scoring experiments carried out in four animals pretreated with the antithrombotic combination heparin and ticlopidine showed marked reduction of both 111In-platelet activity (0.01 +/- 0.03 ml vs. 0.55 +/- 0.49 ml; p less than 0.01) and thrombotic occlusion of the microcirculation (10.8 +/- 7.4 units vs. 34.8 +/- 2.8 units; p less than 0.01) in the right corpus striatum following 3 hours of MCA occlusion. In separate control experiments 111In-labeled autologous platelets were infused after the 3 hour period of right MCA occlusion and subsequent balloon deflation in two animals; no focus of 111In-platelet activity was demonstrated in fixed whole brain.(ABSTRACT TRUNCATED AT 400 WORDS)
We have used a photochemical reaction in vivo to induce reproducible thrombosis leading to cerebral infarction in rats. After the intravenous injection of rose bengal, a potent photosensitizing dye, an ischemic lesion was formed by irradiating the left parietal convexity of the exposed skull for 20 minutes with green light (560 nm) from a filtered xenon arc lamp. Animals were allowed to survive from 30 minutes to 15 days after irradiation. Early microscopic alterations within the irradiated zone included the formation of thrombotic plugs and adjacent red blood cell stasis within pial and parenchymal vessels. Scanning electron microscopy revealed frequent platelet aggregates adhering to the vascular endothelium, often resulting in vascular occlusion. Carbon-black brain perfusion demonstrated that occlusion of vascular channels progressed after irradiation and was complete within 4 hours. Histopathological examination at 1, 5, and 15 days revealed that the associated infarct evolved reproducibly through several characteristic stages, including a phase of massive macrophage infiltration. Although cerebral infarction in this model is initiated by thrombosis of small blood vessels, the fact that the main pathological features of stroke are consistently reproduced should permit its use in assessing treatment regimens. Further, the capability of producing infarction in preselected cortical regions may facilitate the study of behavioral, functional, and structural consequences of acute and chronic stroke.
When the human blood coagulation and fibrinolytic systems are activated thrombin cleaves fibrinopeptide A (FPA) and plasmin cleaves b beta1 leads to 42 from fibrin(ogen). Elevated plasma concentrations of FPA and B beta 1 leads to 42 are evidence for enhanced thrombin and plasma activities in plasma. We have determined the plasma concentrations of FPA and B beta 1 leads to 42 in patients who have had thrombotic stroke. Patients who were studied immediately following stroke were found to have greatly elevated plasma FPA and B beta 1 leads to 42 levels, but these decreased to the concentrations found in an apparently healthy age-matched control group 1 month after the infarct. In contrast, the plasma concentrations of the platelet release product beta-thromboglobulin (beta TG) were slightly, but significantly, elevated immediately following the stroke and these did not alter with time after the infarct. It is concluded that following thrombotic stroke increased thrombin and plasmin activities are to be found in plasma. These increased protease activities are probably not directly associated with an increased in vivo platelet release reaction and may be useful in deciding which patients are at risk of reinfarction or stroke progression.
Thrombosis results, in part, from localized accumulation of fibrin, implying an imbalance between its rate of formation and dissolution. Astrup postulated that patency of the vascular system depended on a dynamic equilibrium between constantly active coagulation and fibrinolytic systems. Reviews of this hypothesis have concluded that neither thrombin nor plasmin proteolysis makes a major contribution to fibrinogen turnover in normal individuals and that the hypothesis of a dynamic equilibrium between clotting and lysis remains unproven. Thus, the current view is that plasmin simply serves the function of a fibrinolytic enzyme digesting fibrin in the vascular system. Here, I promote the alternative view that the relative rates of proteolysis of the B beta chain of fibrinogen by thrombin and plasmin determine the occurrence of thrombosis. This view is supported by recent data and can be used to make some readily testable predictions.
The incidence of mass effect indicated by midline shift, and the incidence of hemorrhagic infarction were studied in 952 consecutive CT scans with supratentorial infarcts. Hemorrhagic infarction was found in 5.1% indicating that the incidence of hemorrhagic infarction during life is far less than was suggested from pathological studies. Midline shift was found in 3.6% and, accordingly, the chance of finding a hemorrhagic infarct with mass effect indicated by a shift of the midline would be 0.2% if these 2 phenomena were not related. However, this incidence appeared to be 12 times higher, indicating they are closely related. In addition, the incidence of hemorrhagic infarcts and midline shift were both related to large infarcts with bad outcome. Cardiac emboli were not more prevalent in patients with hemorrhagic infarcts than in a control series, indicating that cardiac emboli do not produce hemorrhagic infarction more often than other causes of cerebral infarction, and that cardiac embolic cause cannot be inferred from hemorrhagic infarction on CT. This probably also applies to carotid emboli. When anticoagulation is considered in patients with cerebral infarction, CT-confirmed hemorrhagic infarction and shift of midline structures as well as decreased consciousness are contraindications to such treatment. In their absence, and in the absence of general contraindications, anticoagulant treatment can safely be started.
Human extrinsic (tissue-type) plasminogen activator (EPA) was highly purified from the culture fluid of a human melanoma cell line, both as a one-chain or as a two-chain molecule. Its specific fibrinolytic effect on human whole blood clots or plasma clots with different degrees of fibrin crosslinking was evaluated in an in vitro system, composed of a 125I-fibrin labeled clot, hanging in circulating human plasma. After infusion of EPA (30 IU per ml over 3 hrs), non-crosslinked clots lysed more extensively (75-100 percent in 5 hrs) than totally-crosslinked clots (50-65 percent), and no difference was found between one-chain or two-chain EPA. The extent of lysis of totally-crosslinked human or animal plasma clots hanging in autologous plasma induced by EPA varied markedly from one species to the other. When 90 IU of EPA were infused over 3 hrs, crosslinked human plasma clots dissolved for over 95 percent within 5 hrs. Under comparable conditions, the degree of lysis was 80 percent in primate plasma (cynomolgus fascicularis), 60 percent in cat and rabbit plasma, 30 percent in dog plasma and only 10 percent in rat plasma. Systemic activation of the fibrinolytic system in the circulating plasmas was minor and dose-dependent in all species, but complete fibrinogen breakdown was not observed in any species following infusion of up to 90 IU EPA per ml plasma.
It is concluded that the human system is more susceptible to EPA induced fibrinolysis than the other animal systems which were investigated, and that even totally-crosslinked clots can be lysed after infusion of EPA.
Postischemic cerebral inflammation may contribute to ischemic cell damage. Intercellular adhesion molecule-1 (ICAM-1) is a glycoprotein expressed on endothelial cells that facilitates leukocyte adhesion. We investigated the effect of administration of an anti-ICAM-1 antibody (1A29) on ischemic cell damage after transient (2-hour) or permanent middle cerebral artery (MCA) occlusion in the Wistar rat.
Groups studied were as follows: (1) transient MCA occlusion: rats were subjected to 2 hours of MCA occlusion, and after 1 hour of reperfusion they were treated with 1A29 (n = 11) or an isotype control antibody (n = 9); and (2) permanent MCA occlusion: rats were treated with 1A29 (n = 9) or an isotype control antibody (n = 7) 2 hours after onset of MCA occlusion. All animals were killed 1 week after onset of ischemia. Brain sections were stained with hematoxylin and eosin for histological evaluation.
Significant reductions (P < .05) in both volume (44%) of the ischemic lesion and weight loss were found in animals subjected to transient MCA occlusion and treated with 1A29 compared with vehicle-treated animals. In contrast, in animals subjected to permanent MCA occlusion the lesion and the temporal profile of body weight were not altered by 1A29 administration.
Ischemic cell damage is promoted by postischemic inflammatory response after 2 hours of transient MCA occlusion, and ischemic cell damage is reduced by administration of an anti-ICAM-1 antibody during reperfusion.
The integrity of the blood-brain barrier may play an important pathophysiological role during postischemic reperfusion. To determine the factors that lead to exacerbation of brain injury by reperfusion, we investigated changes in cerebral blood flow, blood-brain barrier permeability, edema formation, and infarction in permanent or temporary middle cerebral artery occlusion in rats and studied the relation between local cerebral blood flow and blood-brain barrier disruption.
Middle cerebral artery occlusion was performed with the rat suture model, allowing either permanent (6 hours) or temporary occlusion (3 hours of occlusion and 3 hours of reperfusion). We measured brain water, ion contents, and infarct volumes and determined cerebral blood flow using laser Doppler flowmetry and blood-brain barrier permeability with [3H] alpha-aminoisobutyric acid.
During occlusion, cerebral blood flow was reduced to 7% to 15% (permanent) and 10% to 17% (temporary) of the baseline. During 3 hours of reperfusion, it returned to 47% to 80% (lateral cortex) and 78% to 98% (medial cortex) of the baseline. Compared with the contralateral hemisphere, the water content in the ischemic area increased in both permanent and temporary groups (P < .05, P < .01). Both infarct volume and blood-brain barrier disruption were greater in the reperfusion group compared with the permanent occlusion group (P < .05). Blood-brain barrier disruption correlated with cerebral blood flow during reperfusion (P < .05).
These findings demonstrate that brain infarct and blood-brain barrier disruption are exacerbated after reperfusion in this model of focal ischemia. Blood-brain barrier disruption may relate to the degree of cerebral blood flow recovery. Thus, although early reperfusion in focal ischemia may preserve penumbra tissue, late reperfusion may increase the tissue injury.
The effect of moderate whole-body hypothermia (30 degrees C) on transient middle cerebral artery (MCA) occlusion in the rat was evaluated using diffusion- and perfusion-weighted magnetic resonance imaging. Two hours of transient MCA occlusion was induced by intracarotid insertion of a nylon filament under normothermic (n = 14) and hypothermic (n = 7) conditions. Diffusion- and perfusion-weighted imaging were performed before, during, and after focal ischemia from 30 min up to 7 days. In hypothermic animals, scattered neuronal necrosis was localized to select areas of the caudate putamen and the parietal and insular cortex. In contrast, the normothermic ischemic animals exhibited pan-necrosis and infarct encompassing the damaged area. The diffusion and perfusion data measured from caudate putamen indicate that hypothermia causes a significant reduction in the apparent diffusion coefficient of water (ADCw) and CBF values from normothermic control values (p < 0.01). In both normothermic and hypothermic animals after onset of MCA occlusion, ADCw and CBF values in the core of the ischemic region (striatum) significantly declined from the preischemic and homologous contralateral control ADCw and CBF values (p < 0.05). However, ADCw and CBF in the hypothermic group returned toward control more rapidly than in the normothermic group. These results suggest that the protective effect of hypothermia on ischemic cell damage is reflected in the early return of ADCw during reperfusion and the reduction of ischemic cell damage by hypothermia may be mediated by the improved CBF during acute reperfusion.
The efficacy of thrombolytic therapy for treatment of embolic stroke has been a subject of both experimental and clinical examination. The aim of this study was to compare the efficacy, in regard to reduction of volume of ischemic brain, of two different modes of administration (ie, intra-arterial and intravenous) of tissue-type plasminogen activator (TPA) given 30 minutes after experimental embolic stroke in rabbits.
A randomized, blinded, controlled experimental trial was undertaken. Embolic stroke was simulated in rabbits by injecting fragments of autologous arterial thrombus into one internal carotid artery. Thirty minutes after embolization, the rabbits were blindly treated with 2 mg/kg intra-arterial TPA, 2 mg/kg intravenous TPA, or saline (all n = 10). Six hours after embolization the rabbits were killed. The brains were perfused with triphenyltetrazolium chloride and cut into 0.5-cm-thick coronal sections, and the areas of ischemia were measured.
Administration of TPA resulted in a significant reduction in the volume of ischemic cerebral injury (P < .0001): control animals sustained ischemic injury to 20.1 +/- 4.6% (mean +/- SD) of total brain compared with 4.6 +/- 4.1% for animals treated with intra-arterial TPA and 3.4 +/- 2.6% for those treated with intravenous TPA. The difference between intra-arterial and intravenous TPA treatment was not significant (P = .786).
In this rabbit model of embolic stroke, administration of TPA within 30 minutes resulted in a dramatic reduction in the amount of ischemic injury, with equal efficacy for the two modes of administration. These results favor the treatment of acute embolic stroke with intravenous TPA, given the rapidity with which intravenous therapy can be established in the clinical setting.
Postischemic cerebral inflammation may contribute to ischemic cell damage. The CD11b/18 (Mac-1) integrin mediates stimulated neutrophil binding to endothelia. We therefore investigated the effect of administration of an anti-Mac-1 monoclonal antibody on cerebral ischemic cell damage in the rat.
Rats (n = 10) were subjected to 2 hours of middle cerebral artery occlusion; the anti-Mac-1 antibody was administered at a dose of 2 mg/kg i.v. at 1 hour of reperfusion and 1 mg/kg i.v. at 22 hours of reperfusion or an isotype-matched control antibody (n = 10) was administered using the same experimental protocol. Rats were killed at 46 hours of reperfusion, and brain sections were stained with hematoxylin and eosin for histological evaluation. In a separate population of rats given either vehicle (n = 8) or anti-Mac-1 antibodies (n = 9), intraparenchymal neutrophils were measured by means of a myeloperoxidase assay.
The lesion volume was significantly smaller (28%) in the anti-Mac-1 antibody group compared with the vehicle control group (P < .01). Numbers of intraparenchymal polymorphonuclear cells were significantly reduced (P < .05) in the cortex of the anti-Mac-1 antibody group compared with the vehicle control group.
Our data demonstrate that administration of anti-Mac-1 antibody 1 hour after onset of reperfusion results in significant reductions of ischemic cell damage and intraparenchymal neutrophils after transient (2-hour) focal cerebral ischemia in the rat.
The effect of delayed thrombolysis with recombinant tissue plasminogen activator was tested in an embolic stroke model. The carotid territory was embolized in 103 rats with fibrin-rich clots formed and washed in polyethylene tubes. Hemispheric cerebral blood flow before and after embolization was measured by the intra arterial 133Xe injection method. At five delay times, 15-240 min after embolization, 69 animals were treated with tissue plasminogen activator, 20 mg/kg, and 34 animals with saline. Carotid angiography displayed the grade of occlusion of the cerebral arterial supply before and after treatment. Brains were fixed after 2 days, evaluated neuropathologically, and infarct volume measured. Cerebral blood flow was reduced by 56-71% after embolization. Reperfusion induced by thrombolytic therapy was demonstrated by comparing the posttreatment angiography of the pooled five treatment groups to control animals. Thrombolytic therapy significantly reduced the infarct volume and improved the prekill clinical score by up to 2 h of treatment delay, and treatment might have been beneficial even after 4 h delay. Prolonging the delay of treatment increased the infarct volume (p < 0.001, Jonck-heere-Terpstra test). Only a few hemorrhagic complications were observed. Thus, thrombolytic therapy in embolic stroke induced recanalization. The effect on clinical outcome and infarct volume was dependent on delay time.