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Immunosuppression by whole-body irradiation and its effect on oedema in experimental cerebral ischaemia
Acta Neurologica Scandinavica
Abstract
The effect of global immunosuppression by sublethal whole body X-irradiation on the development of cerebral oedema was assessed 24 h after right middle cerebral artery occlusion in the rat. Irradiation produced a significant leukopenia and thrombocytopaenia, and significantly reduced cortical oedema when compared to non-irradiated control animals.
... To explore this concept, we used brain-only proton irradiation in a focal ischemic stroke model in aged rats. We found this model relevant as low dose radiation modulates scar formation and immune system responses [20][21][22][23][24]. Exposure to low radiation doses modulates the synthesis and release the specific growth factors regulating the glial proliferation [20,21]. ...
... We found this model relevant as low dose radiation modulates scar formation and immune system responses [20][21][22][23][24]. Exposure to low radiation doses modulates the synthesis and release the specific growth factors regulating the glial proliferation [20,21]. Whole body radiation pre-conditioning is neuroprotective as it prevents inflammation in stroke models [22][23][24]. In models of axonal injury, a single low dose of total body or lymphoid organ irradiation reduces secondary neuronal degeneration and stimulates T-cell mediated neuroprotection [25,26]. ...
... Strachan et al. reported that 5 Gy of whole body irradiation 7 days prior to MCAO significantly reduced brain edema 24 h post-stroke. They interpreted their results as radiation-induced immunosupression [24]. While no immune competent organs were directly irradiated in our study, the brain irradiation could effect normal post-stroke microglia signaling and following activation of inflammatory cascade mechanisms. ...
Studies have shown that aging is a significant factor in worsening stroke outcomes. While many mechanisms may aggravate brain injury in the elderly, one such potential system may involve increased glial proliferation in the aged stroke patient that could result in increased scar formation. We hypothesized that in aged rats a single brain-only exposure to a low radiation dose prior to focal brain ischemia would reduce glial proliferation and confer a long-term neuroprotective effect.
Brain-only proton irradiation (8 Gy) was performed ten days prior to middle cerebral artery occlusion (MCAO) in aged male rats. Magnetic resonance imaging (MRI) was undertaken in naive, radiation-only (Rad), MCAO, and MCAO+Rad groups at 2, 14 and 28 days post-stroke followed by immunohistochemistry (day 28).
Ischemic lesion volume in MCAO+Rad group was decreased by 50.7% with an accelerated temporal reduction in peri-lesional brain edema and increased water mobility within the ischemic core (39.8%) compared to MCAO-only rats. In the peri-lesional brain region of MCAO+Rad rats there was a decreased scar formation (49%, glial fibrillary acidic protein), brain tissue sclerosis (30%, aquaporin-4) and necrosis/apoptosis (58%, TUNEL positive cells) compared to those in MCAO animals.
In aged animals a single exposure to brain-only radiation prior to focal cerebral ischemia is neuroprotective as it prevents glial hyperproliferation, progressive brain tissue sclerosis and reduces the apoptosis/necrosis in the peri-lesional region. Decreased lesion volume is in agreement with accelerated reduction of brain edema in these animals.
... 30 Although whole-body irradiation induces oxidative stress in the liver, it is inappropriate to investigate radiation-induced hepatic responses because whole-body irradiation may induce immunosuppression, endocrine dysfunction and multi-organ failure, which are risk factors for hepatic damage. 46,47 Therefore, recent studies have employed hepatic irradiation, which seems to be more suitable for investigating radiation-induced liver responses. When SD rats were exposed to an 8 Gy single dose of abdominal irradiation, severe sinusoidal congestion and hemorrhage, dilation of the central vein, degenerated HCs with perinuclear vacuolization and activated KCs were observed in the irradiated livers 36 h after exposure. ...
Although radiotherapy (RT) is used for the treatment of cancers, including liver cancer, radiation-induced liver disease (RILD) has emerged as a major limitation of RT. Radiation-induced toxicities in nontumorous liver tissues are associated with the development of numerous symptoms that may limit the course of therapy or have serious chronic side effects, including late fibrosis. Although the clinical characteristics of RILD patients have been relatively well described, the understanding of RILD pathogenesis has been hampered by a lack of reliable animal models for RILD. Despite efforts to develop suitable experimental animal models for RILD, current animal models rarely present hepatic veno-occlusive disease, the pathological hallmark of human RILD patients, resulting in highly variable results in RILD-related studies. Therefore, we introduce the concept and clinical characteristics of RILD and propose a feasible explanation for RILD pathogenesis. In addition, currently available animal models of RILD are reviewed, focusing on similarities with human RILD and clues to understanding the mechanisms of RILD progression. Based on these findings from RILD research, we present potential therapeutic strategies for RILD and prospects for future RILD studies. Therefore, this review helps broaden our understanding for developing effective treatment strategies for RILD.
... The histopathological changes after ICH are characterized by early astrocytic swelling, activation of microglia, as well as infiltration of monocytes and neutrophils that coincide with edema formation [15,16,[19][20][21][22][23]. Modulation of the inflammatory response by whole body irradiation to deplete circulating leukocytes and platelets in animal models of ICH decreases injury [24,25]. A greater part of proinflammatory genes, e.g. ...
Intracerebral hemorrhage (ICH) is associated with perihematoma inflammation and edema. We have recently shown cell death and a robust activation of the proinflammatory transcription factor, nuclear factor-κB (NF-κB) in brain areas adjacent to the hematoma. Proteasome represents a key component necessary for the activation of NF-κB. The aim of our present study was to examine if selective proteasome inhibition with a clinically relevant agent, PS-519, might influence the ICH pathogenesis, and improve functional outcome. ICH was induced in Sprague–Dawley rats by the double blood injection method. PS-519 was administered intravenously 4 h and 15 min after induction of ICH. Behavioral testing was performed 3, 5, and 7 days later. The animals were sacrificed on day 7, and their brains were evaluated for hemorrhage size and inflammation using immunohistochemistry with antibody to various inflammatory markers. Treatment with PS-519 significantly (p < 0.05) reduced behavioral impairment post-ICH as determined by the footfault test. This effect was not due to difference in ICH volume. The improved functional status of PS-519 treated animals correlated positively (p < 0.01) with reduced expression of astroglial iNOS in areas adjacent to the hemorrhage 7 days post-ICH. No delayed changes in expression of OX-42 and ED-1 (microglia/macrophages marker), or vimentin (intermediate filament; marker of astroglia activation) were detected in animals treated with PS-519. This data suggests that modulation of proteasome-activated processes may represent a strategic target for treatment of ICH in humans.
... According to previously published data, we evidenced that its low immunogenic properties make pMSC perfect candidates for transplantations without HLA-matching (Le Blank, 2003;Prather et al., 2009). The detected immunosuppressive characteristics of pMSC render this cell source as a promising candidate for stroke therapy, given that stroke outcome is significantly determined by immunoreactions (Strachan et al., 1992). The suppression of allogeneic T-cell proliferation is mediated by the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO), a protein expressed by bmMSC and pMSC (Jones et al., 2007). ...
The beneficial effects of bone marrow-derived mesenchymal stromal cell (MSC) administration following experimental stroke have already been described. Despite several promising characteristics, placenta-derived MSC have not been used in models of focal ischemia. The aim of the current study is to investigate the impact of intravenously transplanted placenta-derived MSC on post-stroke recovery. Permanent occlusion of the middle cerebral artery was induced in spontaneously hypertensive rats. MSC were obtained from the human maternal or fetal placenta and intravenously administered after 24 h (single transplantation) or after 8 h and 24 h (dual transplantation). Sensorimotor deficits were quantified for 60 days using the beam walk test and the modified Neurological Severity Score system. Infarct volume was determined in vivo by means of magnetic resonance imaging on days 1, 8, 29 and 60. Astroglial reactivity was semiquantitatively ascertained within a small and a broad region adjacent to the lesion border. The double infusion of placental MSC was superior to single transplantation in the functional tests. However, a significant difference to the control group in all outcome parameters was observed only for maternally derived MSC. These findings suggest that placental tissue constitutes a promising source for experimental stroke therapies.
... , 1989;Vasthare et aI., 1990;Helps and Gorman, 1991). Strachan et al. (1992) reported significant reduction of ischemic brain edema by rendering rats Iympho-and thrombopenic using whole-body irradiation, without affecting PMNL counts. Finally, a reduction in circulating cell mass, through hemodilutional effects, may per se confer neuroprotection in experimental cerebral ischemia (Fischer and Ames, 1972). ...
White blood cells (WBCs) play vital roles in host defense. Recently, increasing interest has been directed toward the question of whether WBCs, particularly polymorphonuclear leukocytes, could also act as mediators of secondary brain damage in the setting of focal and global cerebral ischemia with and without reperfusion. Considerable insight into the importance of WBC-mediated tissue injury has been gained from studies employing antileukocyte interventions in experimental cerebral ischemia. The purpose of this article is to survey the different approaches taken to interfere with WBC inflammatory function. Emphasis is laid on a discussion of the efficacy of these interventions, their effects and side effects on cerebral and systemic parameters, and the power of evidence they provide for identification of WBCs as important factors in cerebral ischemia. The role of WBCs has been investigated in a great variety of global and focal cerebral ischemia models with and without reperfusion, leading to sometimes contradictory results. In the light of currently available data, it seems likely that WBCs contribute to secondary brain damage in the scenario of experimental transient focal cerebral ischemia, if the insult is not too severe.
... Depletion of neutrophils induced by a variety of regimens, including treatment with anti-neutrophil serum (30), vinblastine (43), or mechlorethamine (12,20) demonstrate decreased size of ischemic deficit and edema formation in neutrocytopenic animals. Similar findings have been reported in animals rendered leukocytopenic by whole body irradiation (24,41). One of the main difficulties with these experiments concerns the inability to completely deplete neutrophils. ...
Adhesion and subsequent penetration of leukocytes into central nervous system ischemic tissue proceeds via a coordinated inflammatory mechanism involving adhesion molecules at the blood-endothelium interface. Mammalian hibernation is a state of natural tolerance to severely reduced blood flow-oxygen delivery (i.e., ischemia). Hibernating thirteen-lined ground squirrels were investigated in an attempt to identify factors responsible for regulating this tolerance. Since leukocytopenia is closely associated with entrance into hibernation, the role of leukocyte adhesion to endothelium in this phenomenon was examined. Intercellular adhesion molecule-1 (ICAM-1) is expressed by endothelium and regulates interactions with circulating leukocytes that may result in margination or extravasation. ICAM-1 expression by rat cerebral microvascular endothelial cells (EC) cultured with plasma from hibernating (HP) or nonhibernating (NHP) thirteen-lined ground squirrels was dose dependently increased by HP and, to a lesser extent, by NHP. Treatment of EC with HP coincidentally induced significantly greater increases in monocyte adhesion to EC (37.2%) than were observed with NHP (23.9%). Study of the effects of HP and NHP on monocyte adhesion to EC may identify mechanisms responsible for ischemic tolerance in hibernators and could lead to the development of novel therapeutic approaches to the treatment of stroke.
... In particular, the degree of leukocyte infiltration following focal stroke correlates with the severity of neuronal injury and neurological deficits in animals ( Clark et al., 1994; del Zoppo et al., 1991) and humans (Akopov et al., 1996). Moreover, in focal ischemia models, anti-leukocyte interventions decrease cerebral edema (Strachan et al., 1992), improve cerebral blood flow (Grogaard et al., 1989; Connolly et al., 1996; Ishikawa et al., 2004), and reduce infarct size (Connolly et al., 1996; Chopp et al., 1994; Beech et al., 2001; Xu et al., 2004; Zheng et al., 2004). Finally, adhesion molecule knockout mice consistently exhibit smaller lesion volumes following focal stroke than their wild-type counterparts (Prestigiacomo et al., 1999; Soriano et al., 1996). ...
Cerebrovascular inflammation contributes to secondary brain injury following ischemia. Recent in vitro studies of cell migration and molecular guidance mechanisms have indicated that the Slit family of secreted proteins can exert repellant effects on leukocyte recruitment in response to chemoattractants. Utilizing intravital microscopy, we addressed the role of Slit in modulating leukocyte dynamics in the mouse cortical venular microcirculation in vivo following TNFalpha application or global cerebral ischemia. We also studied whether Slit affected neuronal survival in the mouse global ischemia model as well as in mixed neuronal-glial cultures subjected to oxygen-glucose deprivation. We found that systemically administered Slit significantly attenuated cerebral microvessel leukocyte-endothelial adherence occurring 4 h after TNFalpha and 24 h after global cerebral ischemia. Administration of RoboN, the soluble receptor for Slit, exacerbated the acute chemotactic response to TNFalpha. These findings are indicative of a tonic repellant effect of endogenous Slit in brain under acute proinflammatory conditions. Three days of continuous systemic administration of Slit following global ischemia significantly attenuated the delayed neuronal death of hippocampal CA1 pyramidal cells. Moreover, Slit abrogated neuronal death in mixed neuronal-glial cultures exposed to oxygen-glucose deprivation. The ability of Slit to reduce the recruitment of immune cells to ischemic brain and to provide cytoprotective effects suggests that this protein may serve as a novel anti-inflammatory and neuroprotective target for stroke therapy.
Leukocyte dynamics were evaluated in vivo in rat retinal microcirculation following exposure to lipid hydroperoxide (LHP) in the vitreous. Leukocytes were observed as fluorescent dots in acridine orange digital fluorography. In the LHP-treated eyes, leukocytes rolling along the major retinal veins were observed and the number increased in a dose-dependent manner (1-10 μg of LHP (18:2)). The flux of rolling leukocytes peaked at 6 h after LHP (10-100 μg) injection. No rolling leukocytes were observed in Linoleic acid (LA) (10 μg)-treated or untreated eyes. The number of accumulated leukocytes started to increase at 4 h and peaked at 24 h after LHP (10 μg) injection. This number was significantly higher than that in LA-treated and untreated eyes. Venous dilation was seen from 4 h after LHP injection and became significant at 6 and 24 h as compared with LA-treated and untreated eyes. The effects of polyethylene glycol-superoxide dismutase (PEG-SOD), one of the important enzyme antioxidants, were investigated. PEG-SOD given intravenously 5 min before LHP (10 μg) injection markedly decreased leukocyte rolling at 6 h. The number of accumulated leukocytes in the PEG-SOD-treated rats decreased signifi cantly at 24 h. These results suggest that increased LHP levels in the vitreous enhance leukocyte-endothelium interaction in the retinal microcirculation, which might be attenuated by PEG-SOD. © Springer Science+Business Media, LLC 2012. All rights reserved.
A newly developed selectin ligand inhibitor (SKK-60060) has been shown to block P- and L-selectins in vitro. We examined its inhibition of leukocyte–endothelial interactions in vivo against retinal ischemia-reperfusion injury and protective effects on ischemia-induced retinal damage.Retinal ischemia was induced by temporary ligation of the optic sheath for 60 min in anesthetized pigmented rats. SKK-60060 was administered 5 min before reperfusion and 4, 12, 24 and 48 hr thereafter, and leukocyte dynamics in the retinal microcirculation were evaluated using acridine orange digital fluorography. After 7 days of reperfusion, ischemia-induced retinal damage was also assessed histologically.SKK-60060 treatment suppressed leukocyte rolling during the reperfusion period; their numbers in the SKK-60060-treated rats were reduced by 67.0% (P < 0.01) and 53.2% (P < 0.01) at 12 and 24 hr, respectively. The subsequent leukocyte accumulation was also inhibited in SKK-60060-treated rats; accumulated leukocytes in the SKK-60060-treated rats were reduced by 72.8% (P < 0.01) and 53.4% (P < 0.01) at 12 and 24 hr, respectively. Retinal venous vasodilation in SKK-60060-treated rats were significantly suppressed at each time point (P < 0.05). Histological examination demonstrated protective effects of SKK-60060 on ischemia-induced retinal damage, which were more substantial in the inner retina (P < 0.01).SKK-60060 significantly inhibits the leukocyte rolling along the major retinal veins and their accumulation during the reperfusion period. These results suggest therapeutic potential of SKK-60060 for ischemia-reperfusion injury.
The clinical and experimental management of stroke has not reached the therapeutic success seen in other medical conditions associated with ischemia/reperfusion. In this work, we investigated the effect of a small molecule selectin inhibitor TBC-1269, in animals subjected to global cerebral ischemia (GCI) and controlled hemorrhagic shock (CHS).
Forty-eight male Sprague-Dawley rats weighting between 275 g and 300 g were subjected to a model of GCI and CHS. Three groups of animals were included in this study (n = 16 per group): sham/saline (group 1); GCI/CHS/Saline (group 2); GCI/CHS/TBC-1269 (group 3). Experimental design consisted of bilateral carotid artery occlusion for 20 minutes, and the development of CHS until a mean arterial pressure of 50 mm Hg was reached. At 20 minutes, clamps were released, and resuscitation was achieved with normal saline, and the end point was to attain a mean arterial pressure of 80 mm Hg. Treatment at the beginning of resuscitation included either normal saline (groups 1 and 2) or TBC-1269 (25 mg/kg, group 3). The following indices were evaluated: brain tissue myeloperoxidase, average numbers of ischemic neurons, and 7-day survival.
Brain myeloperoxidase was decreased in animals treated with TBC-1269, although this difference was not statistically significant. Treated animals demonstrated a significant smaller amount of ischemic neurons than the controls. Survival was also improved from 40% in controls to 80% with TBC-1269 treatment. This difference was statistically significant.
The use of a small molecule selectin inhibitor, TBC-1269, had a protective effect in this model of GCI and CHS, as evidenced by decreased numbers of ischemic neurons and improved survival rates.
Tacrolimus, an immunosuppressant agent, has been shown to reduce tissue injury and leukocyte accumulation after transient ischemia. This study was designed to evaluate quantitatively the inhibitory effects of tacrolimus on leukocyte rolling and on subsequent leukocyte accumulation in vivo after transient retinal ischemia and the protective effects of tacrolimus on ischemia-induced neural damage.
Retinal ischemia was induced for 60 minutes in anesthetized pigmented rats by temporary ligation of the optic sheath. Tacrolimus was administered at 10 minutes after ischemic induction. At 4, 12, 24, and 48 hours after reperfusion, leukocyte behavior in the retinal microcirculation was evaluated in vivo with acridine orange digital fluorography. After 7 days of reperfusion, ischemia-induced retinal damage was evaluated histologically.
Treatment with tacrolimus suppressed leukocyte rolling; the maximum number of rolling leukocytes was reduced by 60.1% at 12 hours after reperfusion (P<0.05). In tacrolimus-treated rats, the velocity of rolling leukocytes was significantly faster than in vehicle-treated rats (P<0.01). The subsequent leukocyte accumulation was reduced by 61.6% at 24 hours after reperfusion (P<0.01). Histological examination demonstrated the protective effect of tacrolimus on ischemia-induced retinal damage, which was more substantial in the inner retina (P<0.01).
The present study demonstrated the inhibitory effect of tacrolimus on leukocyte rolling and on subsequent leukocyte accumulation and the therapeutic potency to neural injury after transient retinal ischemia.
Inflammatory responses are thought to play an important role in the exacerbation of neuronal loss following stroke. Leucocyte recruitment following cerebral ischaemia has been demonstrated in experimental animals, and procedures which reduce the entry of leucocytes into the brain reduce neuronal loss and improve aspects of functional recovery in these models. In this study we investigate whether leakage of plasma proteins into the central nervous system (CNS) following ischaemia influences leucocyte adhesion within the parenchyma. Using an in vitro adhesion assay, we demonstrate that the addition of exogenous serum proteins increases macrophage adhesion to CNS tissue. Following permanent middle cerebral artery occlusion (MCAO) in mice, plasma proteins leak into the apparently healthy cortex surrounding the infarcted area. We show that there is increased macrophage adhesion to sections in the border region where endogenous plasma proteins are present within the parenchyma. Using immunohistochemistry, we co-localize plasma protein distribution within the tissue with leucocyte recruitment following MCAO. We show that monocytes, not neutrophils, infiltrate the lesion border where plasma proteins are present in the parenchyma. This distribution is compatible with their contributing to neuropathology, whereas neutrophils are found in clusters in the lesion core. We conclude that leakage of plasma proteins into the brain could influence leucocyte adhesion within the parenchyma. Recruited monocytes may exacerbate neuropathology in situations such as permanent cerebral ischaemia, where disruption of the blood-brain barrier occurs.
The participation of activated leukocytes and subsequent production of chemical mediators has been well accepted in the pathophysiology of hypoxic-ischemic injury. This study was performed to see the effects of leukocytes on hippocampal neuronal damage in transient global ischemia induced by 10-min occlusion of bilateral common carotid arteries (CCAs) with reperfusion for various times, and in complete unilateral ischemia induced by 24-hr ligation of left CCA. Leukopenia was induced by intraperitoneal injection of cyclophosphamide for 4 days. The results showed that hippocampal neuronal damages were worse at 6-hr reperfusion in leukopenic experimental group than in the control group. In comparison, 24-hr and 3-day reperfusion leukopenic groups showed less numbers of damaged neurons and milder changes. The 5-day reperfusion group showed inconsistent changes. Unilateral CCA occlusion showed extensive infarction in 83.3% of gerbils in the control group, compared to 25% of gerbils in the experimental group (p<0.05). These results strongly suggest that the number of peripheral leukocytes were closely related to the development of delayed neuronal damage of hippocampus in transient global ischemia and the incidence of infarction induced by 24-hr unilateral CCA ligation.
Damage to the central nervous system (CNS) leads to cellular changes not only in the affected neurons but also in adjacent glial cells and endothelia, and frequently, to a recruitment of cells of the immune system. These cellular changes form a graded response which is a consistent feature in almost all forms of brain pathology. It appears to reflect an evolutionarily conserved program which plays an important role in the protection against infectious pathogens and the repair of the injured nervous system. Moreover, recent work in mice that are genetically deficient for different cytokines (MCSF, IL1, IL6, TNFalpha, TGFbeta1) has begun to shed light on the molecular signals that regulate this cellular response. Here we will review this work and the insights it provides about the biological function of the neuroglial activation in the injured brain.
Leukocyte dynamics were evaluated in vivo in rat retinal microcirculation following exposure to lipid hydroperoxide (LHP) in the vitreous. Leukocytes were observed as fluorescent dots in acridine orange digital fluorography.
In the LHP-treated eyes, leukocytes rolling along the major retinal veins were observed and the number increased in a dose-dependent manner (1–10 μg of LHP (18:2)). The flux of rolling leukocytes peaked at 6 h after LHP (10–100 μg) injection. No rolling leukocytes were observed in Linoleic acid (LA) (10 μg)-treated or untreated eyes. The number of accumulated leukocytes started to increase at 4 h and peaked at 24 h after LHP (10 μg) injection. This number was significantly higher than that in LA-treated and untreated eyes. Venous dilation was seen from 4 h after LHP injection and became significant at 6 and 24 h as compared with LA-treated and untreated eyes.
The effects of polyethylene glycol-superoxide dismutase (PEG-SOD), one of the important enzyme antioxidants, were investigated. PEG-SOD given intravenously 5 min before LHP (10 μg) injection markedly decreased leukocyte rolling at 6 h. The number of accumulated leukocytes in the PEG-SOD-treated rats decreased significantly at 24 h. These results suggest that increased LHP levels in the vitreous enhance leukocyte–endothelium interaction in the retinal microcirculation, which might be attenuated by PEG-SOD.
The levels of lipid hydroperoxide (LHPs) in vitreous are elevated in a variety of retinal disorders. Recently, we have shown that increased levels of LHPs in the vitreous enhanced leukocyte-endothelium interaction in the retina, which should contribute to the initial disturbance of the retinal microcirculation. Based upon the previous work, the purpose of the present study was to investigate the effect of polyethylene glycol-superoxide dismutase (PEG-SOD), one of the important enzyme antioxidants, on leukocyte-endothelial interaction in the retinal microcirculation under LHP-induced oxidative stress. Male Brown-Norway rats weighing approximately 250 g were used. LHP(18:2) was made from linoleic acid (LA) with lipoxygenase and 10 microg of LHP dissolved in 5 microl of sodium borate buffer (SBB, 0.02 m) was slowly injected into the vitreous using a 33-gauge needle. PEG-SOD (5000 units/kg) was given intravenously 5 min before LHP injection. At 2, 4, 6, 12, 24 and 48 hr after the vitreous injections, we evaluated the number of rolling leukocytes along the major retinal veins and the number of leukocytes that accumulated in the retinal microvasculature with acridine orange digital fluorography. In LHP-treated rats, leukocyte rolling along the major retinal veins was maximal at 6 hr after LHP injection. The number of rolling leukocytes in the PEG-SOD-treated rats was decreased to 5.5% of those in the LHP-treated rats at 6 hr after LHP injection (P<0.01). No rolling leukocytes were observed in either control or vehicle-treated eyes. The number of accumulated leukocytes in LHP-treated eyes started to increase at 12 hr, and peaked at 24 hr which was significantly higher than in both control and vehicle-treated eyes (P<0.01). The number of accumulated leukocytes in the PEG-SOD-treated rats was reduced by 88.0% at 24 hr (P<0.01). Intravenous injection of PEG-SOD significantly inhibited the leukocyte rolling and its accumulation under LHP-induced oxidative stress. These results suggest that PEG-SOD might attenuate various retinal microcirculatory disorders associated with LHP.
Differences in effects on central haemodynamics, organ blood flow, and serum corticosterone were studied in 11 rats anaesthetized with midazolam/fentanyl/fluanisone (MFF) and 11 other rats anaesthetized with sodium pentobarbital. Compared with pentobarbital, MFF reduced aortic blood pressure by 25%, increased heart rate by 20%, and increased cardiac output by 80%. Unlike most tissues, MFF produced a fivefold increase in blood flow to skeletal muscle, and decreased adrenal blood flow compared to pentobarbital anaesthesia. Initial serum corticosterone levels were lower in rats given MFF anaesthesia, indicating better stress protection. This study also indicates that MFF anaesthesia is preferable to pentobarbital because tissue perfusion generally was better preserved.
The anaesthetic effects of a combination of midazolam, a water-soluble benzodiazepine, and fentanyl-fluanisone were assessed in laboratory rodents and rabbits. This combination of drugs produced good surgical anaesthesia in all of the species investigated. An important advantage of the combination was that it could be administered as a single intraperitoneal injection.
A procedure for occluding the stem of the proximal middle cerebral artery of the rat is described. The operation is performed under anaesthesia through a small subtemporal craniectomy. After occlusion, 3 animals were perfused with carbon block and 8 with a FAM fixative (40% formaldehyde, glacial acetic acid, and methanol). The findings were compared with sham-operated animals. Carbon black studies demonstrated an area of impaired perfusion corresponding to the territory of the occluded artery in each animal. Neuropathological studies invariably showed that there was ischaemic brain damage in the cortex and basal ganglia. The frontal cortex was involved in every animal, as was the lateral part of the neostriatum; the sensorimotor and auditory cortex were involved in most animals, whereas the occipital cortex and medial striatum were involved only infrequently. The damage produced by ischaemia could be readily distinguished from the small local lesion seen at the surgical site in sham-operated animals. The ability to produce a consistent focal ischaemic lesion in the rodent brain provides a technical approach that is sufficiently reproducible to enable investigation of the pathophysiology of ischaemia using recently developed autoradiographic and neurochemical methods.
Granulocytopaenic rats (pretreated with vinblastine) developed brain oedema in tissue surrounding an experimental intracerebral mass 4 hours after the insult in the same way as controls. (Mean S.G. of the ipsilateral caudate nucleus of vinblastine treated and control rats = 1.0467 ± 0.0015 and 1.0464 ± 0.0056 respectively. Mean S.G. on the contralateral sides = 1.0600 ± 0.0033 and 1.0588 ± 0.0031 respectively). This suggests that brain oedema formation in this model after 4 hours occurs independently of the numbers of circulating granulocytes.
CT is the first method of examination that enables exact differential diagnosis of the cerebral infarct. The CT image alone allows differen-tiation between ischemic infarct and hemorrhage. The aim of our in-vestigation was to analyze development and regression of the ischemic infarct and to work out diagnostic and differential diagnostic criteria.
The relationship between increase in water content in ischemic brain and levels of regional blood flow has been studied in 11 primates. Flows were recorded using the method of hydrogen (2-minute) clearance, from a total of 128 electrodes in cortex and white matter, and a gradation of ischemia was produced by middle cerebral occlusion transorbitally. The flows were reduced in the area of densest ischemia from control levels of 12.0 +/- 12.0 ml/100g/min to 7.0 +/- 5.4 ml/100g/min, with lesser decreases over the remainder of the ischemic hemisphere. Water content was measured in cortex and white matter, in regions topographically related to those of flow measurements, by densitometric assessment using precalibrated kerosene/bromobenzine columns. The average water content of cortex in regions remote from ischemia was 797.4 +/- 5.8 mg/gm and in white matter 708.5 +/- 8.2 mg/gm. Significant increases in water content (comparing corresponding regions of the two hemispheres) of up to 11.4 +/- 7.5 mg/gm were demonstrated in the most ischemic cortical areas. A gradient of water increase was evident in the ischemic hemisphere, increases water content being greatest in the opercular zone and least in the parasagittal area. Significant differences in white matter water content between the 2 hemispheres were demonstrated only in the most densely ischemic areas in the current experiments where ischemia was limited to 93 +/- 20 mins in the 11 animals without reperfusion. The relationship between ischemic density and water content increase showed that significant increases in water content occurred in regions where terminal flows had been below 20 ml/100g/min, indicating that accumulation of water in ischemic brain begins at flow values comparable to those associated with the failure of synaptic transmission, higher than those associated with failure of the ionic pump of the cell. Possible pathophysiological mechanisms are discussed.
A classification of brain edema is provided as well as an extensive review of the animal models from which we have derived most of the basic information we have about the formation and resolution of edema. The clinical aspects of cerebral edema in stroke are discussed and also modern methods for identifying cerebral edema in the human. Attention is given to computed tomography and enhanced CT and advances in their application to this condition. Treatment of cerebral edema in the stroke patient using glycerol, dextran 40, mannitol, steroids, and other drugs is discussed and the need pointed out for controlled clinical trials of the therapeutic effectiveness of these agents.
The genesis of ischemic cerebral edema is multi-factorial and the relative importance of these many factors varies throughout the lesion and with time. The initial event is intracellular edema which may be followed by extracellular edema. This situation precludes any simple treatment, particularly because the favorable alteration of one factor might be detrimental to another.
A simple method was developed for the laboratory preparation of gradient columns of specific gravity used in measurement of brain-tissue water. By this automated technique, virtually linear and repeatable density gradients were obtained from which values of tissue specific gravity could be determined. The specific gravity of both solid and fresh cortex and white matter from adult cats was measured and converted to units of percent water per gram tissue using conversion factors derived for this purpose and applicable to studies of brain edema.
Unilateral cerebrovascular occlusion was produced in heparinized rats within 60 seconds after an injection of sodium arachidonate (in doses exceeding 0.45 milligram per kilogram) into the carotid artery. Electroencephalographic activity over the affected cerebral hemisphere was suppressed, and cerebral blood flow decreased by half. Microscopic examination revealed complete obstruction of the hemispheric microcirculation by platelet aggregates.
A lesion simulating intracerebral haemorrhage was produced in the right caudate nucleus of rats immunosuppressed with whole body or regional irradiation. Whole body irradiation produced significant leucopaenia and thrombocytopaenia and conferred protection against cerebral ischaemia and oedema when compared to nonirradiated control animals. Local radiation to the head or torso did not confer protection.
The role of white blood cells in acute central nervous system disorders, such as stroke or traumatic injury is poorly understood. In this experimental study the effect of neutropenia on posttraumatic brain oedema was investigated. Polyclonal antiserum against polymorphonuclear leukocytes was used to induce neutropenia. Control animals received normal serum or no treatment at all. Hemispheric swelling and brain water content after cryo-injury to the exposed left cerebral hemisphere was gravimetrically assessed, and by the wet weight-dry weight method. Administration of anti-neutrophil serum led to a significant reduction of circulating neutrophils. In untreated animals or in rats receiving normal serum, hemispheric swelling of the brain was 7.26 ± 0.35% or 8.52 ± 0.26%, respectively. Neutropenia induced by anti-neutrophil serum resulted in a significant increase in hemispheric swelling to 11.44 ± 0.84% (p < 0.001). It may be noted, however, that the enhancement of brain swelling by neutropenia was not obvious when comparing the water contents of the affected hemispheres of animals subjected to cold injury with and without anti-neutrophil serum.
The enhancement of brain oedema by neutropenia suggests a protective function of neutrophils against extravasation in the presence of a broken blood-brain barrier.
The effect of short and long-term therapy with aspirin (50 mg/day) on platelet alpha granule secretion was studied in 11 healthy controls and 57 patients suffering from transient cerebral ischemic attacks (TIA) with and without accompanying diabetes and hypertension. Plasma levels of beta-thromboglobulin (beta-TG) and platelet factor 4 (PF 4) were measured as indicators of platelet alpha granule secretion. beta-TG and PF 4 levels were increased following cerebral ischemia. Aspirin treatment failed to suppress plasma levels of both proteins when measured a month and then a year after initiation of treatment. Therefore, these proteins may be poor indicators of platelet inhibition by aspirin.
We studied platelet function in whole blood, a situation that better reflects the in vivo state, from 85 patients with acute ischemic stroke and from 19 healthy controls. Patients receiving no antithrombotic drugs demonstrated increased platelet dense body secretion without an associated increase in platelet aggregation, thus raising the possibility that dense body secretion may be of separate importance in cerebral infarction. Our results also suggest that dense body secretion may occur independently of aggregation. Heparin and heparin plus warfarin were ineffective in reducing the high level of dense body secretion seen in acute cerebral infarction, whereas treatment with aspirin plus dipyridamole inhibited both dense body secretion and platelet aggregation. It seems worthwhile to investigate the usefulness of antiplatelet drugs in the treatment of acute ischemic stroke wherein clinical outcome is correlated with the extent of suppression of platelet dense body secretion.
The intradermal injection in rat skin of washed, thrombin-activated platelets produces an increase in vascular permeability, the intensity of which increments with the platelet concentration. Pretreatment of the recipient animals with serotonergic antagonists, including the specific 5-HT2 receptor blocker ketanserin, potently inhibits the platelet-mediated and the 5-HT-induced vascular defect. Amine depletion of platelets or skin tissues with reserpine reduces the response to platelets. Platelet prostanoid and lipoxygenase derivatives play no major role in the vascular response to platelet. The permeability increase induced by exogenous 5-HT and by activated platelets is reduced by alpha 1-adrenergic stimulation with noradrenaline or phenylephrine and by beta 2-stimulation with terbutaline or isoprenaline, and is potentiated by adenosine; this points to a modulation of permeability by blood flow changes and to a direct beta-adrenergic effect at the endothelial cell membrane. This study demonstrates a predominant role for 5-HT in the platelet-mediated vascular permeability increase in a sensitive species like the rat.
In an anesthetized canine model in which ischemia was induced by incremental air embolism, 16 animals were exposed to 1 hr of ischemia and monitored for 10 min (n = 4), 60 min (n = 6), or 240 min (n = 6). Fourteen animals were observed for corresponding periods without being subjected to ischemia 70 min (n = 4), 120 min (n = 4), or 300 min (n = 6). Autologous granulocytes were labeled with 111In and reinfused just before ischemia. At the conclusion of each experiment, a 14C-iodoantipyrine autoradiographic blood flow study was performed. Granulocyte accumulation measured by gamma scintigraphy (cpm/gm) occurred in the injured hemisphere of ischemic animals at 60 min in anterior brain segments and at 240 min in anterior, middle, and posterior segments. By means of a double-label autoradiography technique, clustering of punctate granulocyte images was detected in regions of low flow or heterogeneous flow in half of the animals at both 60 min and 240 min postischemia. Granulocyte clustering did not occur in the autoradiograms of nonischemic animals. The results implicate granulocyte participation in the acute phase of ischemic brain injury and signal a convergence of hemostatic and inflammatory processes during the immediate postischemic period.
The present study was undertaken to analyze the roles of brain cations and of the blood-brain barrier (BBB) to albumin in the development of ischemic brain edema. Using the rat middle cerebral artery (MCA) occlusion model, changes in the brain water, sodium, and potassium contents were followed for a period of seven days. The permeability of the BBB to proteins was also followed by 125I-albumin transfer from the blood into the brain. A significant edema developed as early as three hours after MCA occlusion. This progressed rapidly to reach a maximum on the third day, gradually regressing thereafter. The increase in the brain water contents showed a parallel time course to the increase in the sodium and decrease in the potassium contents. A significant increase in the BBB permeability to albumin occurred 72 hours after MCA occlusion. However, there was no correlation between the brain water content and BBB permeability to albumin in the hemispheres studied 72 hours after MCA occlusion. The correlation between the brain water and sodium contents was not clear during the first six hours, but became highly significant thereafter. The data suggest that an increase in the BBB permeability to sodium occurred 12-48 hours after MCA occlusion, which, together with an antecedent intracellular shift of sodium, resulted in a massive influx of water and sodium into the brain. The BBB permeability change to sodium, not to proteins, seems to play a predominant role in the pathogenesis underlying ischemic brain edema.
The circulating white blood cells of patients with brain infarction were labelled in vitro with Indium-111 tropolonate; the cells were reinjected to study the inflammatory process by gamma camera imaging. Eight patients with acute cerebral ischemic infarct were studied during the first two weeks after the onset of neurological symptoms. In seven cases a well defined area of increased radioactivity was revealed in the infarcted hemisphere indicating active migration and tracking of labelled leukocytes in cerebral infarct. This method allows monitoring of the cellular inflammatory response in human cerebral infarcts and adds another imaging technique.
Various studies indicate that after a latent interval, a progressive focal deterioration of microvascular perfusion can develop in zones of acute damage to the central nervous system (CNS). In order to obtain reliable information concerning the possibility of platelet participation in this phenomenon, platelet deposition was investigated by means of 111Indium-labeled platelets, and compared with local cerebral blood flow (CBF) and cortical sensory evoked response (CSER). Studies were performed with a model of focal ischemia in which small volumes of air were injected selectively into one internal carotid artery of dogs anesthetized with alpha chloralose throughout the experiment. The ischemic period was followed by a 10-min, 60-min or 4-hr recovery period. The main result was a prominent and diffuse accumulation of platelets in zones of low CBF in dogs subjected to 4 hr recovery.
Water content was measured in samples of brain obtained from cats 4 hr to 20 days after occlusion of one middle cerebral artery. Samples were categorized as nonischemic (from hemispheres opposite occluded arteries), ischemic, or infarcted, and as predominantly gray or white matter. When compared with samples from cats with sham operations, water content of each of the 6 types of tissue (including those from nonischemic hemispheres) was increased at 4 hr to 3 days after occlusion. The increase was maximal at 2 days. At all times, water content was greatest in infarcted tissue and least in nonischemic tissue, but from 4 to 20 days, differences were slight. Thus, unilateral experimental cerebral ischemia causes bilateral cerebral edema that is transient and more pronounced in infarcted and ischemic regions.
All cases of acute supratentorial cerebral infarction which came to postmortem examination over the past 10 years at the Philadelphia General Hospital were reviewed. Of a total of 353 such cases, 45 showed severe brain swelling.
Seventy-eight percent of these 45 patients died within seven days of the acute infarction. The rapidly fatal outcome appeared to be directly related to the acute brain swelling with transtentorial herniation and brain-stem edema or hemorrhage.
In those patients who survived longer than one week following onset of the ictus (22%), clinical and anatomic findings suggested that increased intracranial pressure probably did not develop until later in the course of the illness. A second massive infarct was probably superimposed upon an earlier one and produced the acute brain swelling noted at the time of postmortem examination. Complicating visceral diseases were more common in this group and seemed to contribute to death in the majority of these patients.
Although it has been established that a large cerebral infarct may produce sufficient brain swelling and simulate an acutely expanding lesion, this is not widely recognized as playing an important part in determining the patient's clinical outcome.
The role of increased intracranial pressure resulting from acute brain swelling should not be overlooked in the management of patients with severe strokes.
Accumulation of polymorphonuclear neutrophils during the acute inflammatory response may exacerbate tissue injury through the release of activated oxygen products or proteolytic enzymes or both. To assess the role of neutrophils in acute myocardial infarction, circulating neutrophil levels in dogs were reduced by 77 +/- 2% (mean +/- SEM) by administering rabbit antiserum to dog neutrophils. Acute myocardial infarction was induced in open-chest anesthetized dogs by 90 minutes of left circumflex coronary artery occlusion followed by 6 hours of reperfusion. Dogs treated with neutrophil antiserum (n = 8) developed myocardial infarcts that were an average of 43% smaller than infarcts in dogs treated with nonimmune rabbit serum (n = 7) (27.0 +/- 4.5% vs 47.1% +/- 7.5% of the area at risk, p less than 0.05). In a saline-treated control group (n = 8), infarct size was 48.0 +/- 4.7% of the area at risk, a value not significantly different from that of the nonimmune serum group but significantly greater than that in the neutrophil antiserum dogs (p less than 0.05). There were no major hemodynamic differences between groups. Histopathologic examination revealed that infarcted myocardium from dogs given saline or treated with nonimmune serum had a substantial neutrophilic infiltrate, which was virtually absent in infarcted tissue from dogs treated with neutrophil antiserum. These observations suggest that neutrophil accumulation in response to myocardial ischemia may be responsible for a substantial portion of the irreversible myocardial injury resulting from temporary coronary artery occlusion.
A quantitative autoradiographic technique that utilizes carbon-14-aminoisobutyric acid (14C-AIB) as a tracer was used to study alterations in cerebral microvascular permeability in 15 rats. Five were "sham-operated" controls and 10 underwent microsurgical, unilateral occlusion of the proximal middle cerebral artery (MCA). Histological changes indicative of focal cerebral ischemia were observed in only the latter 10 animals. These changes were confined to tissue normally perfused by the occluded MCA. After MCA occlusion, five animals were also subjected to transient halothane-induced hypotension (mean arterial blood pressure 50 mm Hg) for 20 to 30 minutes. Only in these five animals were blood-to-brain transfer constants (ki) significantly increased (by approximately 100%) at 4 hours after MCA occlusion. The topographical distribution of this alteration in cerebral microvascular permeability corresponded closely with the histological changes. Neither proximal MCA occlusion nor halothane-induced hypotension alone was associated with any focal or diffuse increase in ki after 4 hours.
The microgravimetric technique and the drying-weighing method for the determination of brain water content are analyzed and compared. A new method has been devised for the automatic production of the gradient column. For gravimetry, tissue samples weighing more than 30 mg have proven adequate for measurement. Specific gravity (SG) should be determined as early as 1 minute after tissue is inserted into the gradient column. Calculations of cerebral blood volume (CBV) from changes in SG of both brain tissue and intravascular perfusate have shown that the SG of brain tissue is considerably influenced by changes in CBV. This is because the SG of blood is higher than that of brain tissue, and may lead to a decrease of SG of about 0.002 in anemic cortex and of 0.001 in anemic white matter, which will simulate a false increase in tissue volume as water of 4% and 2%, respectively. This methodological error may be relevant when the early stages of ischemic brain edema development are studied. Water content of brain tissue can also be determined with acceptable accuracy by vacuum freeze-drying samples of brain tissue weighing about 100 mg. In contrast to cortex, white matter shows a wide range of individual and regional differences in water content. Thus, conclusions on the presence of brain edema drawn from tissue water determinations should always be subjected to cautious analysis and criticism.
Quanti-tative assessment of early brain damage in a rat model of focal cerebral ischaemia
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An improved gravimetric measure of cerebral edema
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Hemostasis and thrombosis. Basic prin-ciples and clinical practice Platelet mediated vascular permeability in the rat: a predom-inant role for 5-hydroxytryptamine
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Focal cerebral ischemia in the rat: 1: description of technique and early neuropathological consequences following middle cerebral artery occlusion Use of specific gravity in the measurement of cerebral edema The determination of brain water content: mi-crogravimetry versus drying-weighing method
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Effect of neutrophil monoclonal antibody (MoAb 60.3) on infarct volume following transient focal ce-rebral ischemia in cats
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The effect of radiation at the tissue level. In: Biological effects of irradiation
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COGGEL JG. The effect of radiation at the tissue level. In: Biological effects of irradiation. London: Wykeham, 197 1.
Massive cerebral infarction with severe brain swelling: a clinicopathological study Poly-morphonuclear leukocytic accumulation in brain regions with low blood flow during the early postischemic period Reduction of the extent of ischemic myocardial injury by neutrophil depletion in the dog
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Imaging of leukocyte infiltration in human cerebral infarcts Lymphocytes within the infarct area in human brain
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Postischemic antagonism of neutrophil adherence has an acute therapeutic effect on functional recovery in the deteriorating stroke model in rabbits
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LINDSBERG PJ, S I R ~ N A-L, FEUERSTEIN GZ, HALLEN-BECK JM. Postischemic antagonism of neutrophil adherence has an acute therapeutic effect on functional recovery in the deteriorating stroke model in rabbits. J Cereb Blood Flow Metab 1991: 11: Suppl. 2: S754.
Quantitative assessment of early brain damage in a rat model of focal cerebral ischaemia
- Osborne
Postischemic antagonism of neutrophil adherence has an acute therapeutic effect on functional recovery in the deteriorating stroke model in rabbits
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Effect of neutrophil monoclonal antibody (MoAb 60.3) on infarct volume following transient focal cerebral ischemia in cats
- Takeshima
An improved gravimetric measure of cerebral edema
- Marmarou
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Cerebro-vascular permeability following middle cerebral artery occlusion in the rat. The effect of halothane-induced hypotension
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