James Peeling

Intracerebral hemorrhage induces macrophage activation and matrix metalloproteinases

Intracerebral hemorrhage (ICH) is characterized by parenchymal hematoma formation with surrounding inflammation. Matrix metalloproteinases (MMPs) have been implicated in the pathogenesis of neurological diseases defined by inflammation and cell death. To investigate the expression profile and pathogenic aspects of MMPs in ICH, we examined MMP expression in vivo using a collagenase‐induced rat model of ICH. ICH increased brain MMP‐2, ‐3, ‐7, and ‐9 mRNA levels relative to sham‐injected (control) animals in the vicinity of the hematoma, but MMP‐12 (macrophage metalloelastase) was the most highly induced MMP (>80‐fold). Immunohistochemistry showed MMP‐12 to be localized in activated monocytoid cells surrounding the hematoma. In vitro studies showed that thrombin, released during ICH, induced MMP‐12 expression in monocytoid cells, which was reduced by minocycline application. Similarly, in vivo minocycline treatment significantly reduced MMP‐12 levels in brain. Neuropathological studies disclosed marked glial activation and apoptosis after ICH that was reduced by minocycline treatment. Neurobehavioral outcomes also were improved with minocycline treatment compared with untreated ICH controls. Thus, select MMPs exhibit increased expression after ICH, whereas minocycline is neuroprotective after ICH by suppressing monocytoid cell activation and downregulating MMP‐12 expression.Ann Neurol 2003;53:731–742

Experimental Intracerebral Hemorrhage in Rats Magnetic Resonance Imaging and Histopathological Correlates

BACKGROUND AND PURPOSE Intracerebral hemorrhage is associated with a considerable proportion of strokes and head injuries. The mechanism of brain cell injury associated with hemorrhage may be different from that due to pure ischemia. Therefore, it is essential that models of intracerebral hemorrhage be developed and well characterized. The purpose of this study was to obtain high-field MR images of rat brain at progressive times after induction of intracerebral hemorrhage and to correlate the images with behavior and histological evolution. METHODS Intracerebral hemorrhage was induced in rats by injection of bacterial collagenase and heparin into the caudate nucleus. Histopathological changes and corresponding MR images were studied from 30 minutes to 3 weeks after injection. Behavioral changes were also followed for 3 weeks. RESULTS Histological correlation showed that MR is capable of resolving the accumulation and degeneration of the hematoma, a centripetal wave of neutrophils infiltrating from the surrounding tissue beginning at 12 hours, and centripetal invasion of macrophages beginning at 48 hours. Widespread white matter edema was clearly evident on MR images for 1 week after the hemorrhage. Medium-sized striatal neurons were lost in the tissue surrounding the hematoma. Behavioral improvement was rapid during resolution of the edema but incomplete at 3 weeks. CONCLUSIONS MR images correlate very well with histological changes in this experimental model of intracerebral hemorrhage and can therefore be used to follow changes due to drug treatments in vivo. The intense neutrophilic response to this lesion may contribute to neuronal injury at the periphery of the hematoma.

An analysis of four different methods of producing focal cerebral ischemia with endothelin-1 in the rat

Endothelin-1 (ET-1), a potent vasoconstrictor, reduces local blood flow to levels that produce ischemic injury when injected directly into brain tissue. The purpose of this study was to compare 4 different methods of inducing focal ischemia with ET-1: (1) topical application to the forelimb motor region of the cortex, (2) intracerebral injection into the forelimb motor region of the cortex, (3) a combination of intracortical and intrastriatal injections and 4. injection of ET-1 adjacent to the middle cerebral artery (MCA). We examined the effect of delivery method and dose of ET-1 on lesion size, inter-animal variability and behavioral outcome on 3 separate tests of motor function and limb preference. We calculated success rate as the percentage of animals that survived surgery and developed a significant impairment (>20% decrease in performance post-surgery) in the staircase-reaching test. All 4 methods produced similar deficits in the staircase, balance beam, and cylinder tests, but the application of ET-1 adjacent to the MCA, though widely used, provided the lowest success rate. The combined cortical and striatal ET-1 produced a high success rate and consequently we examined cerebral blood flow (CBF), the apparent diffusion coefficient (ADC) and T2-weighted magnetic resonance imaging (MRI) changes for this model. We found that infarct volume measured using T2-weighted MRI correlated with histological measurements and that ADC and CBF together predicted which areas will suffer permanent injury. The combined cortical and striatal injection model offers a number of advantages for studies of recovery of function.

Intraventricular injection of human immunodeficiency virus type 1 (HIV-1) Tat protein causes inflammation, gliosis, apoptosis, and ventricular enlargement

To determine the role of the Tat protein of the human immunodeficiency virus type 1 (HIV-1) in the pathogenesis of HIV-1 associated dementia, recombinant Tat was injected intraventricularly as a single or repeated dose into male Sprague- Dawley rats. Histopathological evaluation showed an initial infiltration of neutrophils one day after Tat injection, followed by macrophages and lymphocytes by 7 days. Tat-injected brains also exhibited astrocytosis, apoptotic cells, and ventricular enlargement 7 days following the last injection. Nuclear magnetic resonance spectroscopic analysis of tissue extracts of hippocampi from Tat-injected rats showed a decrease in the glutamate/g aminobutyric acid ratio. We conclude that the transient extracellular exposure of the central nervous system to Tat protein of HIV can cause a cascade of events leading to the influx of inflammatory cells, glial cell activation, and neurotoxicity.

Antisense Oligodeoxynucleotide Inhibition of Tumor Necrosis Factor-α Expression Is Neuroprotective After Intracerebral Hemorrhage

Background and Purpose— Tumor necrosis factor-&agr; (TNF-&agr;) expression is increased in brain after cerebral ischemia, although little is known about its abundance and role in intracerebral hemorrhage (ICH). A TNF-&agr;–specific antisense oligodeoxynucleotide (ORF4-PE) was used to study the extent to which TNF-&agr; expression influenced neurobehavioral outcomes and brain damage in a collagenase-induced ICH model in rat. Methods— Male Sprague-Dawley rats were anesthetized, and ICH was induced by intrastriatal administration of heparin and collagenase. Immediately before or 3 hours after ICH induction, ORF4-PE was administered directly into the site of ICH. TNF-&agr; mRNA and protein levels were measured by reverse transcriptase–polymerase chain reaction and immunoblot analyses. Cell death was measured by terminal deoxynucleotidyl transferase–mediated uridine 5′triphosphate-biotin nick end labeling (TUNEL). Neurobehavioral deficits were measured for 4 weeks after ICH. Results— ICH induction (n=6) elevated TNF-&agr; mRNA and protein levels (P <0.01) at 24 hours after the onset of injury compared with sham controls (n=6). Immunohistochemical labeling indicated that ICH was accompanied by elevated expression of TNF-&agr; in neutrophils, macrophages, and microglia. Administration of ORF4-PE (2.0 nmol) directly into striatal parenchyma, 15 minutes before (n=4) or 3 hours after (n=6) ICH, decreased levels of TNF-&agr; mRNA (P <0.001) and protein (P <0.01) in the brain tissue surrounding the hematoma compared with animals treated with saline alone (n=6). Mean±SEM striatal cell death (cells per high-powered field) was also reduced in animals receiving ORF4-PE (34.1±5.0) compared with the saline-treated ICH group (80.3±7.50) (P <0.001). ORF4-PE treatment improved neurobehavioral deficits observed at 24 hours (P <0.001) after induction of ICH (n=6) compared with the untreated ICH group (n=6). This improvement was maintained at 28 days after hemorrhage induction (P <0.001). Conclusions— These results indicate a pathogenic role for TNF-&agr; during ICH and demonstrate that reducing TNF-&agr; expression using antisense oligodeoxynucleotides is neuroprotective.

Adenosine A2A receptor activation reduces proinflammatory events and decreases cell death following intracerebral hemorrhage

The ubiquitous neuromodulator adenosine inhibits the production of several proinflammatory cytokines through activation of specific cell‐surface adenosine receptors. We demonstrated recently that antisense oligonucleotides to tumor necrosis factor‐α (TNF‐α) are neuroprotective in a rat model of intracerebral hemorrhage. Therefore, we hypothesized that activation of adenosine receptors would provide protection against intracerebral hemorrhage‐induced TNF‐α production and inflammatory events. In vitro experiments showed that adenosine A1, A2A, and A3 receptor subtypes were present on U937 cells, and activation of these subtypes inhibited TNF‐α production with a rank order of A2A >> A1 > A3. Prolonged treatment of U937 cells with the A2A receptor agonist 2‐p‐(carboxyethyl)phenethylamino‐5′‐N‐ethylcarboxamidoadenosine hydrochloride (CGS 21680) desensitized adenosine A2A, A1, and A3 receptors. CGS 21680 administration directly into the striatum immediately prior to the induction of intracerebral hemorrhage inhibited TNF‐α mRNA and, 24 hours following induction, reduced parenchymal neutrophil infiltration (p < 0.001) and TUNEL‐positive cells (p < 0.002) within and bordering the hematoma. These results suggest that pharmacological strategies targeting A2A receptors may provide effective inhibition of acute neurotoxic proinflammatory events that occur following intracerebral hemorrhage.

Efficacy of disodium 4-[(tert-butylimino)methyl]benzene-1,3-disulfonate N-oxide (NXY-059), a free radical trapping agent, in a rat model of hemorrhagic stroke.

Because free radical mechanisms may contribute to brain injury in hemorrhagic stroke, the effect of the free radical trapping agent disodium 4-[(tert-butylimino)methyl]benzene-1,3-disulfonate N-oxide (NXY-059) was investigated on outcome following intracerebral hemorrhage (ICH) in rat. ICH was induced in 20 adult rats by infusion of collagenase into the caudate-putamen. Thirty minutes later rats were treated with NXY-059 (50 mg/kg subcutaneous plus 8.8 mg/kg/h for 3 days subcutaneous delivered via implanted osmotic pumps) or saline (equivalent volumes). Magnetic resonance imaging 24 h after ICH confirmed that the hemorrhage was uniform in the two groups, and subsequent imaging at 7 and 42 days post-ICH showed that the hematoma resolved similarly in the two groups. Behavioral testing on days 1, 3, 7, 14, and 21 after ICH showed that rats treated with NXY-059 had significantly decreased neurological impairment at all times. Deficits in skilled forelimb use 4-5 weeks post-ICH, and in striatal function 6 weeks post-ICH, were not reduced by treatment with NXY-059. Treatment with NXY-059 significantly reduced the neutrophil infiltrate observed 48 h post-hemorrhage in the vicinity of the hematoma, and the number of TUNEL-positive cells 48 h post-hemorrhage at the hematoma margin. However, by 6 weeks there were no differences in neuronal densities in treated and control rats.

Ischemia-induced cellular redistribution of the astrocytic gap junctional protein connexin43 in rat brain

The distribution and levels of the astrocytic gap junction protein, connexin43 (Cx43) was analyzed in various regions of brain as a function of time after neuronal loss and consequent reactive gliosis induced by bilateral carotid occlusion in rats. In the striatum 2 days after induction of ischemia, immunostaining intensity for Cx43 increased in animals exhibiting mild to moderate striatal damage, whereas areas of reduced staining surrounded by elevated levels of Cx43 immunoreactivity were observed in animals with severe ischemic damage. Immunolabelling of glial cell bodies was evident in ischemic, but not normal, striatum. Similar, though less dramatic, changes were seen at 7 days post-ischemia. Compared with the fine punctate pattern of Cx43 staining seen in normal striatum, ischemic striatal areas contained large aggregates of punctate profiles. In the hippocampus, increased immunostaining was seen at 2 and 7 days post-ischemia and, unlike normal hippocampus, neurons in the CA3 pyramidal cell layer were surrounded by a network of Cx43-immunoreactive puncta at the latter survival time. Immuno-EM analysis of ischemic tissue revealed numerous immunolabelled gap junctions among astrocytic processes in the vicinity of degenerating neurons and elevated levels of intracellular Cx43 immunoreactivity in astrocytic processes and cell bodies. No differences in protein levels or phosphorylation states of Cx43 were detected in either hippocampus or striatum by Western blot analyses of ischemic and control tissue. These results suggest that astrocytes respond to an ischemic insult by reorganizing their gap junctions, that the qualitative nature of their response is dependent on the severity of neuronal damage or loss, and that a pool of Cx43 normally undetectable by immunohistochemistry may contribute to the ischemia-induced elevations of immunolabelling for this protein.

A serial MR study of cerebral blood flow changes and lesion development following endothelin-1-induced ischemia in rats

The vasoconstrictive peptide endothelin‐1 (ET‐1) has been used previously to transiently occlude the middle cerebral artery (MCA) in rats. However, the duration of the resulting reduction in cerebral blood flow (CBF) and the reperfusion characteristics are poorly understood. In this study perfusion and T2‐weighted MRI were used together with histology to characterize the cerebral perfusion dynamics and lesion development following ET‐1 injection. Twenty‐two rats received an intracerebral injection of ET‐1 adjacent to the MCA. CBF was reduced to 30–50% of control levels, and a significant reduction persisted for 16 h in the cortex and 7 h in the striatum. The lesion size measured by T2‐weighted imaging at 48 h correlated with the final infarct size measured by histology at 7 d. The sustained reduction in CBF and the gradual development of the ischemic lesion resemble human stroke evolution, suggesting that this model may be useful for evaluating therapeutic agents, particularly when treatment is delayed. Magn Reson Med 46:827–830, 2001.

Protective effects of free radical inhibitors in intracerebral hemorrhage in rat

Iron compounds formed in the degradation of a hematoma can accelerate the formation of free radicals in adjacent ischemic or hypoperfused tissue. The purpose of this study was to examine the efficacy of compounds that quench free radicals in improving the outcome in rats with experimental intracerebral hemorrhage. Intracerebral hemorrhage was induced in rats by injection of bacterial collagenase and heparin into the caudate nucleus. Rats were treated with alpha-tocopherol plus ascorbic acid starting before hemorrhage, or with dimethylthiourea or alpha-phenyl-N-tert-butyl nitrone starting 2 h after hemorrhage, with treatment continued for 10 days after induction of hemorrhage. Outcome was assessed by behavioral analyses, magnetic resonance imaging, and histopathology. A trend towards behavioral improvement was found for rats treated with alpha-tocopherol/ascorbic acid, while behavior was significantly improved following intracerebral hemorrhage in rats treated with dimethylthiourea or alpha-phenyl-N-tert-butyl nitrone. These results suggest that free radicals may play a role in the development of brain injury following intracerebral hemorrhage, and that compounds that interrupt the free radical cascade may improve outcome. However, treatment did not significantly affect edema, resolution of the hematoma, or neuronal injury in tissue adjacent to the hemorrhage.