Experimental Intracerebral Hemorrhage Model Research Articles

Spontaneous Intracerebral Hemorrhage during Acute and Chronic Hypertension in Mice

Oxidative stress and matrix metalloproteinases (MMPs) contribute to hemorrhagic transformation after ischemic stroke and brain injury after intracerebral hemorrhage (ICH). The goal of this study was to develop a new model of spontaneous ICH, based on the hypothesis that acute, superimposed on chronic, hypertension produces ICH. We hypothesized that increases in angiotensin II (AngII)-mediated oxidative stress and activation of MMPs are associated with, and may precede, spontaneous ICH during hypertension. In C57BL/6 mice, chronic hypertension was produced with AngII infusion and an inhibitor of nitric oxide synthase. During chronic hypertension, mice with acute hypertension from injections of AngII developed ICH. Oxidative stress and MMP levels increased in the brain even before developing ICH. Active MMPs colocalized with a marker of oxidative stress, especially on cerebral vessels that appeared to lead toward regions with ICH. Incidence of ICH and levels of oxidative stress and MMP-9 were greater in mice with acute hypertension produced by AngII than by norepinephrine. In summary, we have developed an experimental model of ICH during hypertension that may facilitate studies in genetically altered mice. We speculate that acute hypertension, especially when induced by AngII, may be critical in spontaneous ICH during chronic hypertension, possibly through oxidative stress and MMP-9.

AβPP/APLP2 Family of Kunitz Serine Proteinase Inhibitors Regulate Cerebral Thrombosis

The amyloid beta-protein precursor (AbetaPP) is best recognized as the precursor to the Abeta peptide that accumulates in the brains of patients with Alzheimer's disease, but less is known about its physiological functions. Isoforms of AbetaPP that contain a Kunitz-type serine proteinase inhibitor (KPI) domain are expressed in brain and, outside the CNS, in circulating blood platelets. Recently, we showed that KPI-containing forms of AbetaPP regulates cerebral thrombosis in vivo (Xu et al., 2005, 2007). Amyloid precursor like protein-2 (APLP2), a closely related homolog to AbetaPP, also possesses a highly conserved KPI domain. Virtually nothing is known of its function. Here, we show that APLP2 also regulates cerebral thrombosis risk. Recombinant purified KPI domains of AbetaPP and APLP2 both inhibit the plasma clotting in vitro. In a carotid artery thrombosis model, both AbetaPP(-/-) and APLP2(-/-) mice exhibit similar significantly shorter times to vessel occlusion compared with wild-type mice indicating a prothrombotic phenotype. Similarly, in an experimental model of intracerebral hemorrhage, both AbetaPP(-/-) and APLP2(-/-) mice produce significantly smaller hematomas with reduced brain hemoglobin content compared with wild-type mice. Together, these results indicate that AbetaPP and APLP2 share overlapping anticoagulant functions with regard to regulating thrombosis after cerebral vascular injury.

Lipoaspirate-derived adult mesenchymal stem cells improve functional outcome during intracerebral hemorrhage by proliferation of endogenous progenitor cells

Stem cell therapy seems promising in reducing deficits after focal cerebral ischemia. As stroke may result from intracerebral hemorrhage (ICH) in up to 20% we investigated whether human processed lipoaspirate mesenchymal stem cells (PLA-MSC) influence the functional outcome, migration behavior and the activation of endogenous progenitor cells. Experimental ICH was induced by stereotactic administration of collagenase in rats randomly assigned to the control or treatment group. The latter received 3 x 10(6) PLA-MSC by intravenous (i.v.) injection 24h after ICH induction. The outcome was continuously monitored using the RotaRod test over a period of 4 weeks. Morphometric analysis of ICH was performed consecutively by magnetic resonance imaging (MRI) studies and immunohistochemical analysis. The RotaRod test revealed a significant 1.5-fold improvement (p<0.005) in functional outcome for the PLA-MSC treated group after 4 weeks compared to controls. Histological and MRI assessment of lesion size showed no difference between the two groups. Although i.v. injected human cells could not be detected in the post mortem brain, evaluation of the number of endogenous progenitor cells revealed a twofold increase in the treated animals compared to controls. Treatment with PLA-MSC improved the functional outcome significantly in an experimental ICH model. This effect was achieved by stimulation of endogenous progenitor cells rather than integration and differentiation of the infused PLA-MSC.

Addition of intravenous N-methyl-D-aspartate receptor antagonists to local fibrinolytic therapy for the optimal treatment of experimental intracerebral hemorrhages

Fibrinolytic therapy with recombinant tissue plasminogen activator (rtPA) is considered a treatment option in patients with deep-seated intracerebral hemorrhage (ICH). Nevertheless, the results of animal experiments have shown that tPA exerts pleiotropic actions in the brain, including regulation of vasoactivity, amplification of calcium conductance by cleavage of the N-methyl-D-aspartate (NMDA) receptor subunit, and activation of metalloproteinases, which increase excitotoxicity, damage the blood-brain barrier, and worsen edema. The authors investigated whether the noncompetitive NMDA receptor antagonist MK801 can be used as an adjuvant therapy in combination with rtPA to attenuate the unfavorable delayed edema formation and inflammation observed following rtPA therapy in an experimental porcine model of ICH. Twenty pigs were used in this study; MK801 (0.3 mg/kg) was administered to each pig intravenously immediately after hematoma induction and on the 1st and 3rd day after hematoma induction. Ten of the 20 pigs were randomly assigned to fibrinolytic therapy with rtPA (MK801-tPA group), whereas in the remaining 10 control animals (MK801 group) the hematomas were allowed to follow their natural courses of resorption. The extent of edema formation was evaluated using magnetic resonance (MR) imaging volumetry on Days 0, 4, and 10 after hematoma induction and was compared with histopathological changes found at necropsy. The mean edema volumes in these two groups were also compared with that in the group of nine pigs examined in a preceding experimental series, in which the animals' hematomas were only treated with rtPA (tPA group). In the 10 animals in the MK801-tPA group, the mean perihematoma edema volume on MR images had not significantly increased by Day 4 (p < 0.08) or Day 10 (p < 0.35) after hematoma induction. In the 10 animals in the MK801 group, the increase in mean perifocal edema size was significant after 4 days (p < 0.001) and nonsignificant after 10 days (p < 0.09). In the nine animals in the tPA group, the mean edema volume significantly increased by Days 4 (p < 0.002) and 10 (p < 0.03). As suggested by the reduction in delayed edema volume and the inflammatory response, MK801 modifies the neurotoxic properties of rtPA but not those of blood degradation products. Possibly, fibrinolytic therapy of ICH is more beneficial if combined with agents such as MK801.

Proteasomal inhibition in intracerebral hemorrhage: Neuroprotective and anti-inflammatory effects of bortezomib

Inflammation is an important pathophysiologic mechanism of injury induced by intracerebral hemorrhage (ICH). The ubiquitin-proteasome system (UPS) regulates the inflammatory responses via the up-regulation of several pro-inflammatory molecules. In this study, we determined that a potent proteasome inhibitor, bortezomib, exerted therapeutic effects in experimental model of ICH. Either bortezomib (0.05, 0.2, 0.5, 1 mg/kg) or vehicle was intravenously administered 2 h after ICH induction. The high doses of bortezomib caused high mortality rates. Bortezomib at 0.2 mg/kg reduced the early hematoma growth and alleviated hematoma volume and brain edema at 3 days after ICH, compared with the ICH-vehicle group. The numbers of myeloperoxidase + neutrophils, Ox42 + microglia, and TUNEL + cells in the perihematomal regions were decreased by bortezomib. Bortezomib induced significant decrements of mRNA expression of TNF-α and IL-6. The production of iNOS and COX2 was also reduced significantly by bortezomib. We concluded that the early treatment with bortezomib induced a reduction in the early hematoma growth and mitigated the development of brain edema, coupled with a marked inhibitory effect on inflammation in ICH.

Local brain hypothermia for neuroprotection in stroke treatment and aneurysm repair

Hypothermia is well known to provide neuroprotection following various brain insults in experimental animals. Two recently completed clinical trials of whole body hypothermia in outof–hospital cardiac arrest patients' demonstrated significantly improved survival rates and neurologic outcomes. These results provide new excitement and encouragement for clinical application of hypothermia in cerebrovascular disease. However, the intensive care challenges and adverse events (e.g. prolonged times to target temperatures, shivering and sedation, pneumonia) during the management of hypothermia, dampen enthusiasm for widespread application especially in elderly stroke patients. In this manuscript, we review recent hypothermia trials for stroke. We describe an alternate approach, i.e. local brain cooling, and discuss this new technique with reference to the extensive literature on the marked efficacy of hypothermia. We describe a new technology, the ChillerPadTM and ChillerStripTM Systems developed by Seacoast Technologies, Inc. (Portsmouth, NH, USA). The latter device has received FDA approval and will be employed in a trial of local hypothermia for cerebral aneurysm repair. We present our experimental findings that profound local hypothermia does not damage cortical neurons. We also report that local hypothermia protects the blood–brain barrier and markedly reduces vasogenic edema development in an experimental intracerebral hemorrhage model. Lastly, we review potential mechanisms through which hypothermia provides blood–brain barrier protection and reduces edema formation. Clearly, hypothermia has a bright future for cerebrovascular disease treatment if brain cooling can be delivered in a manner that does not compromise the patient or the neurosurgical and intensive care settings. Local brain cooling may be just that new treatment approach.

Brain Tissue Oxygen Monitoring in Intracerebral Hemorrhage

Brain tissue oxygen (PbrO2) monitoring is an emerging technique for detection of secondary brain injury in neurocritical care. Although it has been extensively reported in traumatic brain injury and aneurysmal subarachnoid hemorrhage, its use in nontraumatic intracerebral hemorrhage (ICH) has not been well described. We report complementary preliminary studies in a large animal model and in patients that demonstrate the feasibility of PbrO2 monitoring after ICH. To assess early events after ICH, Licox Clark-type oxygen probes were inserted in the bilateral frontal white matter of four anesthetized swine that subsequently underwent right parietal hematoma formation in an experimental model of ICH. Intracranial pressure (ICP) was monitored as well. Seven patients with acute ICH, who were undergoing ICP monitoring as part of standard neurocritical care, had placement of a frontal oxygen probe, with subsequent monitoring for up to 7 days. In the swine ICH model, a rise in ICP early after hematoma formation was accompanied by a decrease in ipsilateral and contralateral PbrO2. Secondary increases in hematoma volume resulted in further decreases in PbrO2 over the first hour after ICH. In patients undergoing oxygen monitoring, low PbrO2 (<15 mmHg) was common. In these patients, changes in FiO2, mean arterial pressure, and cerebral perfusion pressure (but not ICP) predicted subsequent change in PbrO2. Brain tissue oxygen monitoring is feasible in ICH patients, as well as in a swine model of ICH. Translational research that emphasizes complementary information derived from human and animal studies may yield additional insights not available from either alone.

Quantitative analysis of injured, necrotic, and apoptotic cells in a new experimental model of intracerebral hemorrhage.

To develop a new survival model of intracerebral hemorrhage (ICH) in rabbits and study the patterns of cellular injury in different regions 24 hrs after introduction of hematoma. Quantitation and characterization of injured cells in regions adjacent and distant to the hematoma have not been performed. Prospective case-control study. Ten New Zealand rabbits. We introduced ICH in six anesthetized New Zealand rabbits by autologous blood injection under arterial pressure in the deep white matter in the frontal lobe. Hematoxylin and eosin staining was performed in six animals with ICH after 24 hrs to quantify intact, injured, and necrotic cells in regions proximal and distant to the hematoma, and the results were compared with four control animals. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining was performed to quantify apoptotic cells in specified regions in five animals with ICH, and the results were compared with four control animals. All cell counts were performed by one investigator who used 100x oil emersion microscopy. The presence of localized hematoma was confirmed in all six animals with blood infusion. Compared with controls, animals with ICH had a significantly higher proportion of swollen cells in both the inner (55.9% +/- 3.0% vs. 26.8% +/- 1.7%; p < .05) and the outer (59.8% +/- 4.6% vs. 27.7% +/- 4.5%; p < .05) rim of the perihematoma region. A small proportion of shrunken dark cells were observed in both the inner (4.0% +/- 1.5%) and the outer (3.6% +/- 1.0%) rim of the perihematoma region. The remaining cells were considered morphologically intact. A large proportion of cells trapped within the matrix of the hematoma were either shrunken dark cells (48.8% +/- 16.4%) or swollen (38.8% +/- 15.1%). In the TUNEL-stained sections, a high burden of apoptotic cells was observed in the matrix of the hematoma (17.5 +/- 6.3 cells per high power field) but not in the perihematoma regions (less than two cells per high power field). A reproducible model of ICH in rabbits is described. At 24 hrs, the perihematoma region contains relatively large proportions of morphologically intact or reversibly injured (swollen) cells, suggesting the possibility of an extended window for therapeutic intervention.

Citicoline treatment for experimental intracerebral hemorrhage in mice.

Citicoline sodium (cytidine-5'-diphosphocholine) has been shown previously to reduce ischemic injury in focal central nervous system models. Intracerebral hemorrhage (ICH) appears to be associated with an area of edema and ischemic injury surrounding the hematoma that may be reduced by neuroprotective therapy. The present study was designed to test whether treatment with citicoline reduces ischemic injury and improves functional neurological outcome in an experimental model of ICH. In 68 Swiss albino mice (26 to 36 g), ICH was induced by collagenase injection into the caudate nucleus. Animals were randomized to receive either: citicoline 500 mg/kg or saline IP prior to collagenase and at 24 and 48 hours. Animals were rated on a 28-point neurological scale and sacrificed at 54 hours. The brains were sectioned, and the volume of hematoma, total lesion, and surrounding ischemic injury was determined. In terms of functional outcome, animals treated with citicoline had improved neurological outcome scores compared with placebo-treated animals: 10.4+/-2.0 versus 12.1+/-2.4 (P<0.01). Regarding ischemic injury, although there was no difference in the underlying hematoma volumes, animals treated with citicoline had a smaller surrounding volume of ischemic injury than placebo-treated animals: citicoline, 13.8+/-5.8 mm3 (10.8+/-4.3% of hemisphere); placebo, 17.0+/-7.1 mm3 (13.3+/-5. 1%) (P<0.05). In this animal model of ICH, treatment with citicoline significantly improved functional outcome and reduced the volume of ischemic injury surrounding the hematoma. This study supports a potential role for citicoline in clinical ICH treatment.

Induction of NADPH-diaphorase activity in the forebrain in a model of intracerebral hemorrhage and its inhibition by the traditional Chinese medicine complex Nao Yi An.

Induction of NADPH-diaphorase (NDP) activity in the rat cerebral cortex was studied after autologous blood injection into the internal capsule as experimental model of intracerebral hemorrhage. The potential inhibitory effect on NDP induction by Nao Yi An (NYA), a complex derived from materials of animal and plant origin used in the treatment of intracerebral hemorrhage in traditional Chinese medicine, was also investigated. In animals without therapeutic treatment 2 and 4 days after injection of autologous blood, NDP activity was highly induced in pyramidal neurons in the neocortex, piriform, and entorhinal cortices, in astrocytes and in phagocytes in the hematoma and the area surrounding it, as well as in the subcortical white matter, and in endothelial cells in both the cortex and subcortical white matter bilaterally. Oral administration of NYA failed to inhibit NDP induction in endothelial cells but demonstrated a strong inhibitory effect on NDP activity induced in pyramidal neurons and astrocytes. NDP induction in phagocytes was also inhibited by the administration of NYA. Altogether the present results suggest that intracerebral hemorrhage in the internal capsule may induce nitric oxide synthase activity in different cell populations in the cortex and that administration of NYA can selectively inhibit such induction and, thus, potentially play a neuroprotective role.

Experimental intracerebral hemorrhage in rats. Magnetic resonance imaging and histopathological correlates.

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. 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. 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. 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.