Treatment Of Focal Cerebral Ischemia Research Articles

An Optimal Animal Model of Ischemic Stroke Established by Digital Subtraction Angiography-Guided Autologous Thrombi in Cynomolgus Monkeys.

ObjectiveIschemic stroke seriously threatens human health, characterized by the high rates of incidence, disability, and death. Developing a reliable animal model that mimics most of the features of stroke is critical for pathological studies and clinical research. In this study, we aimed to establish and examine a model of middle cerebral artery occlusion (MCAO) guided by digital subtraction angiography (DSA) in cynomolgus monkeys.Materials and MethodsIn this study, 15 adult male cynomolgus monkeys were enrolled. Under the guidance of DSA, a MCAO model was established by injecting an autologous venous clot into the middle cerebral artery (MCA) via femoral artery catheter. Thrombolytic therapy with alteplase (rt-PA) was given to eight of these monkeys at 3 h after the occlusion. Blood test and imaging examination, such as computed tomography angiography (CTA), CT perfusion (CTP), brain magnetic resonance imaging (MRI), and brain magnetic resonance angiography (MRA), were performed after the operation to identify the post-infarction changes. The behavioral performance of cynomolgus monkeys was continuously observed for 7 days after operation. The animals were eunthanized on the 8th day after operation, and then the brain tissues of monkeys were taken for triphenyltetrazolium chloride (TTC) staining.ResultsAmong the 15 cynomolgus monkeys, 12 of them were successfully modeled, as confirmed by the imaging findings and staining assessment. One monkey died of brain hernia resulted from intracranial hemorrhage confirmed by necropsy. DSA, CTA, and MRA indicated the presence of an arterial occlusion. CTP and MRI showed acute focal cerebral ischemia. TTC staining revealed infarct lesions formed in the brain tissues.ConclusionOur study may provide an optimal non-human primate model for an in-depth study of the pathogenesis and treatment of focal cerebral ischemia.

Transplantation of Human Amniotic Mesenchymal in the Treatment of Focal Cerebral Ischemia

This study examined the potential of human amniotic mesenchymal (hAMSC) transplantation in repairing neurological deficits in an experimental focal cerebral ischemia model. Following isolation of hAMSC, growth characteristics and surface antigen expression was observed. Butylated hydroxyanisole (BHA) was used to induce the cultured cells into neuron-like cells, identified by immunocytochemistry. The suture model was used to induce focal cerebral ischemia in the rats, which were subsequently randomly divided into experimental and control groups for treatment with BrdU-labeled hAMSCs or PBS, respectively. Neurological deficits were assessed after transplantation using the Neurological Severity Scores, Beam Balance Test, and Elevated Body Swing Test. Eight weeks later rat brain tissue was processed for hematoxylin-eosin staining and BrdU immunohistochemistry, and survival and spatial distribution of transplanted hAMSCs. hAMSCs proliferated in vitro, and neuron specific enolase expressed in neurons and glial fibrillary acidic protein in astrocytes. The focal ischemia model resulted in varying degrees of left limb hemiplegia accompanied by the right side of Horner’s sign. When examined 1, 3, 6 and 8 weeks later, significant recovery in the neurological behavior was detected in the rats treated with the hAMSC transplantation compared to the control (P<0.01). BrdU-labeled hAMSCs were concentrated near the graft site and surrounding areas, in some cases migrating towards the ischemic lesion. Local gliosis and lymphocytic infiltration were not detected. hAMSC exhibit great potential for proliferation, and can be induced to differentiate into NSE-expressing neuron-like cells following treatment with BHA. Moreover, hAMSC transplantation may improve neurological symptoms after focal cerebral ischemia.

Transplantation of Adipose Tissue-Derived Stem Cells for Treatment of Focal Cerebral Ischemia

The neurological functional disabilities caused by cerebral infarction significantly deteriorate life quality and increase the medical and socio-economic costs. Although some molecular agents show potential in acting against the pathological mechanisms in animal studies, none has been proven effective for cerebral ischemia treatment in human patients. New treatment strategy needs to be developed. Stem cell therapy is promising for neural regeneration and thus become one of the current trends. More evidence has shown stem cells, such as embryonic stem cells (ESCs), skeletal muscle satellite cells and mesenchymal stem cells, to be useful in tissue repair and regeneration. However all these stem cells mentioned above have limitations. Adipose tissue-derived stem cells (ADSCs) are an alternative autologous stem cell source for the characters as abundant, easy to obtain, immunological and ethic problem free. So far, this treatment strategy has been rarely adopted on ischemic brain injury. In this study, we investigated the transplantation effects of rat ADSCs for the treatment of cerebral ischemia in rats. ADSCs were isolated from rat adipose tissue and then induced to initiate neural differentiation. Following neural induction, ADSCs developed neural morphology and displayed molecular expression of Nestin, MAP2 and GFAP. We evaluate the neurobehavioral function, infarct volume and cell properties as apoptosis, survival, migration, proliferation, differentiation and immunogenicity. Treatment with i-ADSCs (induction from ADSCs) results in better functional recovery and more reduction in hemispheric atrophy then without i-ADSCs in other groups. Our study demonstrates that i-ADSCs therapy is promising in stroke treatment and finally leads to an efficacious therapeutic modalities for much better outcome in clinical patients.

Filtrate of Phellinus linteus Broth Culture Reduces Infarct Size Significantly in a Rat Model of Permanent Focal Cerebral Ischemia

Phellinus linteus, a natural growing mushroom, has been known to exhibit anti-tumor, anti-inflammatory, anti-allergic and anti-oxidant effects. Aiming to exploit the neuroprotective effects of P. linteus, we evaluated its effects on infarct volume reduction in a rat model of focal cerebral ischemia. Male Sprague-Dawley rats were subjected to right middle cerebral artery occlusion. Filtrate of P. linteus broth culture (various doses), fractionated filtrate (based on molecular weight) or control medium was administered intraperitoneally to rats before or after ischemia induction. Rats were killed at 24 h after the stroke surgery. Cortical and caudoputaminal infarct volumes were determined separately using an image analysis program following staining with 2,3,5-triphenyltetrazolium chloride. Significant cortical infarct volume reductions were found in the pre-treatment groups (30 and 60 minutes before onset of cerebral ischemia) compared with the control group, showing dose dependence. Posttreatment (30 minutes after ischemic onset) also significantly reduced cortical infarct volume. Furthermore, the higher molecular weight (≥12 000) fraction of the culture filtrate was more effective compared with the lower molecular weight fraction. The present findings suggest that P. linteus may be a new promising approach for the treatment of focal cerebral ischemia, with the additional benefit of a wide therapeutic time window since significant infarct volume reduction is obtained by administration even after the ischemic event. Our finding that the higher molecular weight fraction of the P. linteus culture filtrate demonstrated more prominent effect may provide a clue to identify the neuroprotective substances and mechanisms.

UCF‐101, A Novel Omi/HtrA2 Inhibitor, Protects Against Cerebral Ischemia/Reperfusion Injury in Rats

The aim of this study was to investigate the therapeutic efficacy and neuroprotective mechanisms of UCF-101, a novel Omi/HtrA2 inhibitor, following ischemia/reperfusion brain injury. Male Wistar rats were subjected to 2 hr of middle cerebral artery occlusion followed by reperfusion. Animals were divided into 3 groups: sham, vehicle-treated ischemia/reperfusion, and UCF-101 treatment. In the UCF-101 treatment group, rats were intraperitoneally administered UCF-101 (1.5 micromol/kg) 10 min prior to reperfusion. The rats were evaluated for neurological deficits, and brain infarct volume was assessed by 2,3,5-triphenyl tetrazolium chloride. TUNEL staining was utilized to evaluate the amount of apoptosis. In addition, expressions of protein caspase-8, caspase-3, FasL, and FLIP were examined by Western blot analysis. Results demonstrated that UCF-101 treatment significantly decreased cerebral infarct size by about 16.27% (P < 0.05) and also improved neurological behavior. TUNEL staining revealed that UCF-101 treatment significantly reduced TUNEL-positive cells in the cerebral cortex. Furthermore, the upregulation in the expression of FasL and the cleavage products of active caspase-8 and caspase-3 induced by ischemia was attenuated in mice treated with UCF-101, whereas upregulation of FLIP levels was increased. The present results demonstrated that UCF-101 protects against cerebral ischemia/reperfusion injury in mice. UCF-101 provided neuroprotection in vivo, and this was correlated with regulation of Fas-mediated apoptotic proteins. Taken together, the use of UCF-101 is a potent, neuroprotective factor for the treatment of focal cerebral ischemia.

Matrix Metalloproteinase 2 (MMP2) and MMP9 Secreted by Erythropoietin-Activated Endothelial Cells Promote Neural Progenitor Cell Migration

We investigated the hypothesis that endothelial cells activated by erythropoietin (EPO) promote the migration of neuroblasts. This hypothesis is based on observations in vivo that treatment of focal cerebral ischemia with EPO enhances the migration of neuroblasts to the ischemic boundary, a site containing activated endothelial cells and angiogenic microvasculature. To model the microenvironment within the ischemic boundary zone, we used a coculture system of mouse brain endothelial cells (MBECs) and neural progenitor cells derived from the subventricular zone of the adult mouse. Treatment of MBECs with recombinant human EPO (rhEPO) significantly increased secretion of matrix metalloproteinase 2 (MMP2) and MMP9. rhEPO-treated MBEC supernatant as conditioned medium significantly increased the migration of neural progenitor cells. Application of an MMP inhibitor abolished the supernatant-enhanced migration. Incubation of neurospheres alone with rhEPO failed to increase progenitor cell migration. rhEPO activated phosphatidylinositol 3-kinase/Akt (PI3K/Akt) and extracellular signal-regulated kinase (ERK1/2) in MBECs. Selective inhibition of the PI3K/Akt and ERK1/2 pathways significantly attenuated the rhEPO-induced MMP2 and MMP9, which suppressed neural progenitor cell migration promoted by the rhEPO-activated MBECs. Collectively, our data show that rhEPO-activated endothelial cells enhance neural progenitor cell migration by secreting MMP2 and MMP9 via the PI3K/Akt and ERK1/2 signaling pathways. These data demonstrate that activated endothelial cells can promote neural progenitor cell migration, and provide insight into the molecular mechanisms underlying the attraction of newly generated neurons to injured areas in brain.

Hypothermia and Cerebral Ischemia: Mechanisms and Clinical Applications.

Wake Forest University School of Medicine, Winston-Salem, North Carolina. petrozza@wfubmc.eduHypothermia and Cerebral Ischemia: Mechanisms and Clinical Applications. By Carolina M. Maier, Ph.D., and Gary K. Steinberg, M.D., Ph.D. Totowa, New Jersey, Humana Press, 2003. Pages: 192. Price: $99.50.Hypothermia as a protective therapy and a treatment for cerebral ischemia in multiple settings has a long and storied history. Many anesthesiologists and intensivists have seen the prominence, the waning, and now the resurgence of therapeutic hypothermia within their professional lifetimes. Hypothermia and Cerebral Ischemia: Mechanisms and Clinical Applications is intended to provide a comprehensive review of mild hypothermia, including the therapeutic potential of the methodology, limitations, and recent developments in both basic and clinical research.One of the pleasures of reading this book is the evident effort which the editors Carolina A. Maier, Ph.D., and Gary K Steinberg, M.D., Ph.D., both from Stanford University, Stanford, California, expended on organizing the multiauthored text. The initial chapters contain a review of moderate hypothermia in experimental animal models primarily, and the middle chapters discuss current clinical practice and clinical trials. The third section of the book summarizes several studies performed primarily at single European centers in stroke patients. The summative chapter attempts to highlight future directions for the therapeutic applications of moderate hypothermia. Each chapter is organized with a concise historical view of hypothermia in the relevant experimental or clinical context, followed by presentations of important research studies and then a discussion of possible mechanisms of action, with a final concluding section on future directions for research.While the text is multiauthored with several international contributors, repetition of information is kept to a minimum, particularly in the initial chapters discussing experimental models of moderate hypothermia. Evidence of careful editing throughout the text is apparent by references to material or discussion in previous chapters where appropriate. In addition, the book is nicely illustrated with appropriate tables and images from experimental trials, which serve to highlight the teaching points within each chapter.Perhaps because a busy clinician has little time to explore the experimental basis for common therapies, this reader found the initial five chapters of this text to be most enjoyable. The concise discussions of the experimental rationale for moderate hypothermia followed by exploration of its application for the prevention and treatment of focal cerebral ischemia, traumatic brain injury, and postischemic injury were excellent. In contrast, the latter chapters of the text, which highlighted clinical trials and clinical methods of application of moderate hypothermia, were occasionally uneven in the amount of detail provided about clinical trials, research protocols, and patient groups.An obvious issue with a text that attempts to provide a comprehensive review of multiple applications of a technique in diverse groups of patients with many clinical scenarios is the issue of the current relevance of the information. The best asset of this text is its use to provide a foundation from which a clinician can best interpret new data. There are many practitioners, particularly those who specialize in neuroanesthesia and critical care, who are undoubtedly familiar with the myriad of mechanisms now identified as having a role in patient outcome after cerebral ischemia. However, a significant group of clinicians still exists who believe that an individual therapy such as hypothermia or a particular anesthetic might be the next “magic bullet.” Useful reading of the text such as Hypothermia and Cerebral Ischemia: Mechanisms and Clinical Applications at the point where one is initially exposed to the concepts of cerebral protection or neurosurgical anesthesia or professional interest is aroused through a clinical scenario might prove to be extremely valuable in broadening the knowledge base of many practitioners.This eminently readable text would be a valuable edition to the library of anesthesiology trainees who develop an interest in “cerebral protection” as they become familiar with concepts of neurosurgical anesthesia, critical care, and cerebral preservation within the operating room. In addition, this text serves as a strong foundation for those involved in the care of patients with devastating neurosurgical and neurologic disease as well the general practitioner of anesthesiology whose intellectual curiosity propels one to understand the background, limitations, and methodologies of current highly publicized clinical trials using therapeutic moderate hypothermia and other cerebral protection modalities.Wake Forest University School of Medicine, Winston-Salem, North Carolina. petrozza@wfubmc.edu

Treatment of Focal Cerebral Ischemia with Liposomal Nerve Growth Factor

Liposomal nerve growth factor (NGF) was used for the treatment of focal cerebral ischemia in a rat model. Positive charge inducing agents of sphingosine (SP) and stearylamine (S) were formulated in the liposomal NGF. Dose-response of intraventricular injection of liposomal NGF showed significant reduction in infarct volume at the dose of 5 and 10 μ g/rat of NGF. The liposomal NGF formulated with SP or S demonstrated similar results in the reduction of total infarct volume in rats. When we increased the molar ratio of SP and S from 0.15 to 0.3, the infarct volume from rats showed a similar value as that of the control treated with NGF solution. Liposomal NGF was given prior to the development of ischemia. We found that NGF was effective in prevention of neuronal death. The NGF concentrations in brain for liposomal NGF were maintained in a level significantly higher than those for NGF solution. This was attributed to the positively charged liposomal NGF bound effectively in brain ventricle and caused longer retention time than free NGF for localization in brain. Therefore, the effect of liposomal NGF on reduction of infarct volume was significant. We assumed that the transportation of NGF might go through the cerebrospinal fluid pathway throughout the ventricular system and subarachnoid system to cerebral cortex to produce a therapeutic effect on ischemia.

Spinal cord stimulation reducing infarct volume in a model of focal cerebral ischemia in rats.

The authors previously showed that spinal cord stimulation (SCS) increases cerebral blood flow in rats, indicating that this technique may be useful in the treatment of focal cerebral ischemia. In the present study, the neuroprotective potential of SCS in the setting of middle cerebral artery occlusion (MCAO) was investigated. The authors induced permanent, focal cerebral ischemia by using either suture-induced occlusion or direct division of the MCA in Sprague-Dawley rats. Electrical stimulation of the cervical spinal cord was performed during cerebral ischemia. Cerebral blood flow was assessed using both laser Doppler flowmetry (LDF) and quantitative radiotracer analysis. Stroke volumes were analyzed after 6 hours of ischemia. Spinal cord stimulation resulted in a 52.7 +/- 13.3% increase in LDF values (nine animals). Following MCAO, LDF values decreased by 64.1 +/- 3.6% from baseline values (10 animals). Spinal cord stimulation subsequently increased LDF values to 30.9 +/- 13.5% below original baseline values. These findings were corroborated using radiotracer studies. Spinal cord stimulation in the setting of transcranial MCAO significantly reduced stroke volumes as well (from 203 +/- 33 mm3 [control] to 32 +/- 8 mm3 [MCAO plus SCS], seven animals in each group, p < 0.001). Similarly, after suture-induced MCAO, SCS reduced stroke volumes (from 307 +/- 29 mm3 [control] to 78 +/- 22 mm3 [MCAO plus SCS], 10 animals in each group, p < 0.001). A strategy of performing SCS for the prevention of critical ischemia is feasible and may have the potential for the treatment and prevention of stroke.

Role of a synthetic pyrimidine compound, MS-818, in reduction of infarct size and amelioration of sensorimotor dysfunction following permanent focal cerebral ischemia in rats.

A synthetic heterocyclic pyrimidine compound, MS-818 (2-piperadino-6-methyl-5-oxo-5,6-dihydro-(7H) pyrrolo-[3,4-d] pyrimidine maleate) is reported to have a variety of biological activities including neurite outgrowth, astrocyte differentiation, suppression of neuronal apoptosis, regeneration of injured peripheral nerves, fracture repairs, angiogenesis, and superovulation. To be able to explicate the neurotrophic effects of MS-818, the authors evaluated its effect on the reduction of infarct volume and amelioration of sensorimotor dysfunction in a rat model of focal ischemia. Forty male Sprague-Dawley rats were subjected to right middle cerebral artery occlusion and assigned to one of four treatment groups (10 animals in each group). The MS-818 (1, 5, or 10 mg/kg) or phosphate-buffered saline (control group) was administered intraperitoneally at onset of ischemia and again 24 hours later. The rats were killed 48 hours after they underwent surgery to induce stroke, and infarct volume was determined using an image-analysis computer software program following staining with 2,3,5-triphenyltetrazolium chloride. Postischemic neurological deficit and body weight were also assessed. Significant reductions in infarct volume (total and cortical infarction) were found in all the MS-818-treated groups compared with the control group. Furthermore, MS-818 induced significant amelioration of sensorimotor dysfunction, as indicated by the results of forelimb and hindlimb placing tests. The present findings suggest that MS-818, which has a much smaller molecular weight than neurotrophic peptides. represents a new approach to the treatment of focal cerebral ischemia.

Delayed neuroprotective effect of insulin-like growth factor-i after experimental transient focal cerebral ischemia monitored with mri.

Insulin-like growth factor (IGF) treatment has been shown to have trophic and neuroprotective effects in vitro and in vivo in different lesion models. IGF-I has potent neuroprotective effects after hypoxic-ischemic injury and global ischemia. The role of IGF-I in focal cerebral ischemia is only partially understood. Therefore, in the present study, we evaluated, by applying MRI monitoring, whether a clinically relevant systemic administration of IGF-I can achieve a long-lasting neuroprotective effect. Male Wistar rats underwent transient occlusion of the right middle cerebral artery for 1 hour by using the suture occlusion model. Animals then were intraventricularly treated with 33.33 microg IGF-I/d for 3 days (group A, the IGF-I group [n=13]; group B, the placebo group [n=14]) or subcutaneously treated with 200 microg IGF-I/d for 7 days (group D, the IGF-I group [n=10]; group E, the placebo group [n=10]). Groups C and F served as sham-operated controls (n=5 and n=3, respectively). Treatment was begun 30 minutes after occlusion of the middle cerebral artery. Subcutaneously treated animals underwent MRI studies (diffusion-weighted imaging, perfusion imaging, and T2-weighted imaging) beginning 60 minutes after vessel occlusion at 6 hours and at days 1, 2, 5, and 7 after ischemia. The animals were weighed and neurologically assessed daily (rating scale ranged from 0, indicating no deficit, to 5, indicating death). On the third day (intraventricular trial) and on the seventh day (subcutaneous trial), animals were euthanized, and brain sections were stained with triphenyltetrazolium chloride. The mean infarct volume was 52.9+/-25.2 mm(3) in intraventricularly treated animals versus 146.4+/-62.2 mm(3) in control animals (P<0.01) and 42.2+/-17.9 mm(3) in subcutaneously IGF-I-treated animals versus 73.1+/-38.1 mm(3) in control animals (P<0.05). Apparent diffusion coefficient-derived lesion volume at 60 minutes after occlusion was 40.4+/-23.7 mm(3) versus 38.3+/-19.3 mm(3) (P=NS), increased to 168.3+/-49.55 mm(3) versus 105.5+/-33.8 mm(3) (P<0.05) at 24 hours, and then decreased to 55.8+/-30.3 mm(3) versus 23.3+/-20.2 mm(3) (P<0.05) for control and IGF-I-treated animals, respectively. The T2-weighted-derived ischemic lesion volume at 24 hours after occlusion was 236+/-49.2 mm(3) versus 115.9+/-56.8 mm(3) (P<0.05) and decreased to 115.9+/-26.2 mm(3) versus 75.7+/-35.8 mm(3) (P<0.05) at day 7 for control and IGF-I-treated animals, respectively. The relative regional cerebral blood volume was reduced to 50% before reperfusion in all regions of interest except for region of interest 1 (vessel territory of anterior cerebral artery), recovered during reperfusion, but was not different between the control and the growth factor-treated group at any imaging time point. There was no significant difference in weight loss. There was less neurological deficit after ischemia in intraventricularly and subcutaneously IGF-I-treated animals compared with control animals (P<0.05). Continuous treatment with intraventricularly and subcutaneously administered IGF-I achieved a long-lasting neuroprotective effect as early as 24 hours after ischemia as measured by MRI. Therefore, IGF-I may represent a new approach to the treatment of focal cerebral ischemia.

Patency of cerebral microvessels after focal embolic stroke in the rat.

In patients with thrombotic stroke, the occluded artery often reopens over time. This results through a natural dissolution of the occluding material, and fragments of the material may move downstream to obstruct distal arteries. The current study was undertaken to investigate the patency of brain microvessels at varying time intervals after injection of a preformed clot into the right internal carotid artery of rats. Cerebral microvessels in brain sections were visualized using immunohistochemistry for fibronectin (detecting existing microvessels) and Evans blue (visualizing perfused microvessels). The percentage of patent microvessels was calculated as the number of Evans blue-positive microvessels divided by the number of fibronectin-positive microvessels. In normal control animals, results showed that 98% +/- 3% (mean +/- SD) of microvessels in the cortex and 94% +/- 14% in the striatum were patent. In the ischemic animals, immediately after clot injection, microvessels in the cortex and striatum were occluded, mainly in the territory irrigated by the middle cerebral artery. One hour after clot injection, microvessels had reopened in most of the cortex but remained occluded in some portions of the striatum, possibly as a result of downstream movement of fragments formed from the original clot. By 3 hours after clot injection, microvessels in the cortex were patent in all animals, whereas in the striatum microvessels were patent in 50% of the animals. In the other 50%, small striatal perfusion deficits persisted. At 24 hours after clot injection, microvessels were patent in both the cortex and striatum of all animals except one. These findings suggest that intracerebral clots dissolve spontaneously in a relatively short period of time, but that fragments formed from the clot may obstruct more distal blood vessels. It is likely that clot fragments lodge in arteries with lower blood flow and poor collateral perfusion, where they continue to cause ischemia for a longer duration. These results may in part explain the resistance of the striatum to neuroprotective strategies used for the treatment of focal cerebral ischemia.

Neuroprotective effects of progesterone after transient middle cerebral artery occlusion in rat.

Treatment of focal cerebral ischemia in the rat with intraperitoneal administration of progesterone dissolved in dimethyl sulfoxide (DMSO) has demonstrated therapeutic efficacy. In the present study we test whether iv administration of water soluble progesterone 2 h after the onset of middle cerebral artery occlusion provides therapeutic benefit for the treatment of stroke. In addition, we perform a battery of functional tests: rotarod, adhesive-backed somatosensory, and neurological score, as well as a dose-response study. The data indicate that iv administration of progesterone at a dose of 8 mg/kg significantly reduces the volume of cerebral infarction and significantly improves outcome on the array of functional measures employed. Treatment with 4 mg/kg or 32 mg/kg of progesterone failed to provide any therapeutic benefit. Progesterone, a non toxic, clinically employed, pluripotent therapeutic agent which targets both neuroprotective as well as neuroregenerative strategies, may have important therapeutic benefits for the treatment of stroke.

Neutrophil inhibitory factor treatment of focal cerebral ischemia in the rat

The present study was designed to determine whether a hookworm-derived recombinant neutrophil inhibitory factor (rNIF) is neuroprotective when administered after initiation of focal cerebral ischemia in the rat. We measured the rNIF dose–response on cerebral infarct volume, the therapeutic time window, the therapeutic response to permanent ischemia, and whether rNIF treatment delays the maturation of the ischemic lesion (2 days), or reduces cerebral infarct volume at 7 days after middle cerebral artery occlusion (MCAO). MCAO was induced by an insertion of intraluminal 4-0 monofilament nylon suture into internal carotid artery ( n=195). We demonstrate a significant neuroprotective effect of rNIF administration 48 h after MCAO in a dose-dependent fashion when treatment was initiated upon reperfusion after 2 h MCAO and maintained until 48 h after MCAO. The beneficial effect was lost under conditions of permanent MCAO. The therapeutic time window is 4 h after MCAO. Brief treatment (6 h) is not sufficient to provide protection for the final ischemic damage. Continuous treatment with a high dose of rNIF for a long duration (7 days) is necessary to achieve maximum neuroprotection.

Adenosine kinase inhibition protects brain against transient focal ischemia in rats

Endogenous adenosine released locally during cerebral ischemia is neuroprotective, and agents which decrease adenosine inactivation may potentiate its protective effects. The effects of 5′-deoxy-5-iodotubercidin (5′d-5IT), an inhibitor of the adenosine-catabolizing enzyme, adenosine kinase, were studied in male Wistar rats subjected to 2 h of transient middle cerebral artery occlusion. 5′d-5IT or the vehicle (10% DMSO in saline) was administered i.p. 30 min before, and 2 h and 6 h after the induction of middle cerebral artery occlusion. The infarct volume was determined using 2,3,5-triphenyltetrazolium chloride staining 48 h after middle cerebral artery occlusion. The infarct volume was significantly reduced in rats treated with 1.85 mg/kg×3 (57% reduction, P<0.001) or 1.0 mg/kg×3 (34% reduction, P<0.05), but not 0.3 mg/kg×3 5′d-5IT compared to vehicle-treated rats. The reduction of infarct volume was accompanied by a significant improvement in behavioral measures of neurological deficit. These data further support a role of adenosine in neuroprotection and suggest that adenosine kinase inhibition may be a useful approach to the treatment of focal cerebral ischemia.

Treatment of Focal Cerebral Ischemia With Synthetic Oligopeptide Corresponding to Lectin Domain of Selectin

Synthetic oligopeptides with amino acid sequences of the lectin domain of selectin block selectin-mediated cell adhesion in vitro, which may be applied to a therapeutic intervention to attenuate acute inflammatory reactions. To evaluate the efficacy of such treatment against ischemic brain injury, the effects of administering a selectin oligopeptide that selectively blocks selectin-mediated cell adhesion on histological outcome and on cerebral blood flow (CBF) were studied in models of rodent focal cerebral ischemia. Spontaneously hypertensive rats were anesthetized with halothane. Permanent focal cerebral ischemia was induced by tandem left middle cerebral artery (MCA) and common carotid artery (CCA) occlusion. Focal cerebral ischemia with partial reperfusion was introduced by reperfusing the CCA after 2 hours of tandem MCA/CCA occlusion. A synthetic oligopeptide (amino acid residues 23-30 from N terminal) of E-selectin was dissolved in physiological saline and was injected intravenously at a dosage of 2 mg/kg or 10 mg/kg before artery occlusion. Control animals received equivalent volumes of physiological saline or 10 mg/kg of synthetic oligopeptide with a scrambled amino acid sequence. Twenty-four hours after the occlusion, seven coronal brain slices were stained with 2,3,5-triphenyltetrazolium chloride, and the volume of ischemic injury was calculated. In a separate set of animals, regional CBF was monitored with laser-Doppler flowmetry at the dorsolateral cerebral cortex during 2-hour ischemia and 30 minutes after partial reperfusion. The volume of ischemic injury did not differ among groups in permanent ischemia. In ischemia with partial reperfusion, 10 mg/kg selectin oligopeptide, but not the same dosage of scrambled oligopeptide, significantly reduced the volume of ischemic injury (95 +/- 13, 73 +/- 11, 55 +/- 6, and 111 +/- 14 mm3 for saline [n = 11]; 2 mg/kg [n = 10] and 10 mg/kg [n = 16] selectin oligopeptide and 10 mg/kg scrambled oligopeptide [n = 6], respectively; P < .01 by one-way ANOVA followed by the Tukey test). Laser-Doppler flowmetry demonstrated a larger increase in CBF after reperfusion of the CCA in the 10-mg/kg selectin oligopeptide group. Our data demonstrate that administration of a synthetic oligopeptide corresponding to the lectin domain of selectin decreases the size of ischemic injury after transient, but not after permanent, focal cerebral ischemia as evaluated at 24 hours after onset of ischemia. These effects were associated with an improved CBF at the dorsolateral cerebral cortex after partial reperfusion.

Evaluation of delayed treatment of focal cerebral ischemia with three selective kappa-opioid agonists in cats.

The purpose of this study was to determine the therapeutic efficacy of three kappa-opioid agonists used for delayed treatment of experimental focal cerebral ischemia. Forty halothane-anesthetized cats underwent permanent occlusion of the right intracranial internal carotid, middle cerebral, and anterior cerebral arteries via a transorbital, microsurgical approach. Six hours after occlusion, animals received a blinded bolus injection, and a subcutaneous osmotic pump was implanted to provide continuous release for 7 days. The injection and pump contained either saline or one of three kappa-agonists: dynorphin (1-13), U-50,488, or DuP E3800. Survival, neurological function, tissue damage, and brain weight were assessed. As a group, kappa-agonist-treated animals had higher survival (P < .02), less tissue damage (P < .02), and lower brain weight (P < .05) than saline controls. U-50,488 more effectively improved survival (P < .03) than dynorphin (P < .07) or E3800 (P < .07). Each of the three kappa compounds improved tissue damage (dynorphin, P < .02; U-50,488, P < .05; E3800, P < .05). Greater improvement in neurological function was seen after treatment with dynorphin (P < .05) than with U-50,488 (P < .6) or E3800 (P < .7). The only significant reduction in brain weight was seen after dynorphin treatment (P < .01). Compounds that act at the kappa subclass of opiate receptors are effective in increasing survival, improving neurological function, and decreasing tissue damage and edema in a cat model of focal cerebral ischemia. The current study provides support for the benefits of treatment of acute cerebrovascular ischemia with kappa-opioid agonists. The agents may prove to be of superior clinical utility because of efficacy even when administered 6 hours after the onset of stroke.

BW619C89, a glutamate release inhibitor, protects against focal cerebral ischemic damage.

The excitatory amino acid neurotransmitter glutamate is involved in excitotoxic brain injury and neurodegeneration after cerebral ischemia. Therefore, compounds that block the release of glutamate may be useful as cerebroprotective agents. The purpose of this study was to evaluate the cerebroprotective properties of a glutamate release inhibitor, BW619C89. In the studies reported here, the effect of BW619C89 [4-amino-2-(4-methyl-1-piperazinyl)-5-(2,3,5-trichlorophenyl)pyrimidine] on neurotransmitter release (endogenous amino acids, gamma-aminobutyric acid, and acetylcholine) from slices of rat brain cerebral cortex in vitro has been determined. The neuroprotective efficacy of BW619C89 has been evaluated using the middle cerebral artery occlusion model of focal cerebral ischemia in the Fischer 344 rat. In the in vitro studies, BW619C89 inhibited veratrine- (but not potassium-) evoked release of both endogenous glutamate and aspartate from rat cerebral cortex slices with IC50 values of approximately 5 microM. BW619C89 was approximately 10-fold less potent to inhibit veratrine-evoked 3H-gamma-aminobutyric acid release (IC50 = 51 microM), fourfold less potent to inhibit 3H-acetylcholine release (IC50 = 21 microM), and at 10 microM had only weak activity at excitatory amino acid (N-methyl-D-aspartate, kainate, and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) binding sites. When administered intravenously to Fischer 344 rats 5 minutes after permanent middle cerebral artery occlusion, BW619C89 produced marked reductions of both total (cortex and basal ganglia) and cortical infarct volumes. Cortical infarct size was reduced by 20% at a dose of BW619C89 of 5 mg/kg (n = 6, not significant); 43% at 10 mg/kg (n = 8, P < .01); 59% at 20 mg/kg (n = 8, P < .001); 61% at 30 mg/kg (n = 8, P < .001), and 53% at 40 mg/kg (n = 8, P < .001). BW619C89 at doses of 20 and 30 mg/kg also significantly reduced noncortical (basal ganglia) infarct volumes, demonstrating that a proportion of this tissue also appears to be salvageable. Behavioral effects observed were dose related, generally minor, and at doses of 20 mg/kg IV and above consisted of body tremor and mild ataxia lasting approximately 2 hours. These results suggest that glutamate release inhibitors such as BW619C89 may provide an alternative to excitatory amino acid receptor antagonists in the treatment of focal cerebral ischemia and stroke.

Pathophysiology and treatment of focal cerebral ischemia. Part II: Mechanisms of damage and treatment.

The mechanisms that give rise to ischemic brain damage have not been definitively determined, but considerable evidence exists that three major factors are involved: increases in the intercellular cytosolic calcium concentration (Ca++i), acidosis, and production of free radicals. A nonphysiological rise in Ca++i due to a disturbed pump/leak relationship for calcium is believed to cause cell damage by overactivation of lipases and proteases and possibly also of endonucleases, and by alterations of protein phosphorylation, which secondarily affects protein synthesis and genome expression. The severity of this disturbance depends on the density of ischemia. In complete or near-complete ischemia of the cardiac arrest type, pump activity has ceased and the calcium leak is enhanced by the massive release of excitatory amino acids. As a result, multiple calcium channels are opened. This is probably the scenario in the focus of an ischemic lesion due to middle cerebral artery occlusion. Such ischemic tissues can be salvaged only by recirculation, and any brain damage incurred is delayed, suggesting that the calcium transient gives rise to sustained changes in membrane function and metabolism. If the ischemia is less dense, as in the penumbral zone of a focal ischemic lesion, pump failure may be moderate and the leak may be only slightly or intermittently enhanced. These differences in the pump/leak relationship for calcium explain why calcium and glutamate antagonists may lack effect on the cardiac arrest type of ischemia, while decreasing infarct size in focal ischemia. The adverse effects of acidosis may be exerted by several mechanisms. When the ischemia is sustained, acidosis may promote edema formation by inducing Na+ and Cl- accumulation via coupled Na+/H+ and Cl-/HCO3- exchange; however, it may also prevent recovery of mitochondrial metabolism and resumption of H+ extrusion. If the ischemia is transient, pronounced intraischemic acidosis triggers delayed damage characterized by gross edema and seizures. Possibly, this is a result of free-radical formation. If the ischemia is moderate, as in the penumbral zone of a focal ischemic lesion, the effect of acidosis is controversial. In fact, enhanced glucolysis may then be beneficial. Although free radicals have long been assumed to be mediators of ischemic cell death, it is only recently that more substantial evidence of their participation has been produced. It now seems likely that one major target of free radicals is the microvasculature, and that free radicals and other mediators of inflammatory reactions (such as platelet-activating factor) aggravate the ischemic lesion by causing microvascular dysfunction and blood-brain barrier disruption.(ABSTRACT TRUNCATED AT 400 WORDS)

The efficacy of intravenous nimodipine in the treatment of focal cerebral ischemia in a primate model.

The clinical and pathological effects of nimodipine on cerebral infarction were investigated in 12 male baboons. In randomized/blind trials, six animals given intravenous nimodipine (2 micrograms/kg/min load, 1 microgram/kg/min maintenance) for 96 hours starting 50 minutes before 6-hour double-clip occlusion of the middle cerebral artery were compared to 6 control animals. Standardized neurological examinations were performed by examiners blinded to the animals' therapy on Day 7 and Day 14 after stroke. On Day 14 the animals were killed. The brains were studied pathologically, and the relative areas of infarction were quantified. Intracranial pressure was lower in nimodipine-treated animals; however, the range of intracranial pressure values in each group was broad. Two control animals with high intracranial pressure died. There were no deaths among the nimodipine-treated animals. The neurological scores on Days 7 (P less than or equal to 0.01) and 14 (P less than or equal to 0.05) were significantly different between the two groups. The nimodipine-treated animals had less clinical evidence of infarction compared to controls. Nimodipine-treated animals tended to have smaller areas of infarction; however, the difference between the two groups was not statistically significant. The infusion of nimodipine in the treatment of focal cerebral ischemia is safe and does not appear to aggravate the extent of infarction or to exacerbate intracranial hypertension. The clinical neurological evaluations indicate that nimodipine may improve or preserve neurological outcome after stroke.