Intracerebral hemorrhage models in rat: comparing collagenase to blood infusion.

Many therapies have shown promise in preclinical stroke studies, but few benefit patients. A greater understanding of stroke pathophysiology is needed to successfully develop therapies, and this depends on appropriate animal models. The collagenase and blood infusion models of intracerebral hemorrhage (ICH) are widely used; yet, investigators often prefer using one model for a variety of reasons. Thus, we directly compared these to highlight advantages and limitations of each as well as the assessment approach. An ICH was created by infusing blood or bacterial collagenase into the rats' striatum. We matched initial hematoma volume in each model (Experiment 1) and assessed the time course of bleeding (Experiment 2). Functional deficits and the progression of injury were tracked over 6 weeks using behavior, magnetic resonance imaging, and histology (Experiment 3). Despite similar initial hematoma volumes, collagenase-induced ICH resulted in a greater blood-brain barrier breakdown and more damage to the striatum, substantia nigra, white matter, and cortex. Magnetic resonance imaging revealed faster hematoma resolution in the blood model, and little increase in the volume of tissue lost from 1 to 6 weeks. In contrast, tissue loss continued over 4 weeks in the collagenase model. Finally, functional deficits recovered more quickly and completely in the blood model. This study highlights key differences between these models and that neither closely replicates the human condition. Thus, both should be used whenever possible taking into account the significant differences between these models and their limitations. Furthermore, this work illustrates significant weaknesses with several outcome measures.

Executive dysfunction and blockage of brain microvessels in a rat model of vascular cognitive impairment.

Most research focuses on overt stroke caused by blockage of major blood vessels. Less attention has been paid to small vessel disease which gives rise to covert stroke that often leads to vascular cognitive impairment (VCI). One reason for this may be the relative lack of relevant animal models. Herein, we describe, a model of VCI induced in middle-aged Sprague-Dawley rats exposed to a diet high in saturated fats, salt and refined sugar (HFSS). In Experiment 1, rats were fed HFSS and subjected to a small mediodorsal (MD) thalamic stroke with or without concomitant permanent bilateral carotid artery occlusion. MD lesions produce significant executive dysfunction in an attention set-shift task ( p = 0.012). In Experiment 2, rats were exposed to either HFSS or control diet and functional effects assessed. We found significant hypertension ( p = 0.013), blockage of brain microvessels ( p = 0.018) and white matter atrophy ( p = 0.039) in HFSS diet animals. As in Experiment 1, profound, specific set-shifting executive dysfunction was noted ( p = 0.003) following both small MD infarcts (0.332 mm3) and the HFSS diet. In summary, these data describe a middle-aged animal model of VCI that includes clinically relevant metabolic disturbances and small vessel disease and as such may be helpful in developing new cognitive therapies.

Protective effect of minocycline treatment on striatal ischemia.

Minocycline reduces infarct volume measured up to 1 week after focal cerebral ischemia, but it has not been shown that this results in lasting improvement in functional outcome. This study examined behavioral outcome in rats out to 3 weeks after focal ischemia induced by injection of the vasoconstrictor endothelin (ET)-1 (400 pmol in 1 microL of saline) into the striatum. Magnetic resonance imaging confirmed reduced blood flow after administration of ET-1, and was used to determine lesion volumes at 1 and 21 days postischemia. In control rats, intraperitoneal injection of minocycline resulted in plasma levels of 6.6 +/- 2.7 microg mL(-1) between 1 and 8 hours after administration. Based on these results, intraperitoneal minocycline treatment was started either 1 hour before or 3 hours after ET-1 administration, and was repeated daily for 5 days. Outcome, assessed using a composite behavioral deficit score (days 2, 4, 7, 14, and 21) and a test of asymmetric forelimb use (days 7 and 21), was significantly better in both groups of rats treated with minocycline, and the improvement was maintained for the 3-week study period. No differences were found in infarct volumes between groups.

Does thrombin play a role in the pathogenesis of brain damage after periventricular hemorrhage?

Neonatal periventricular hemorrhage (PVH) is a devastating complication of prematurity in the human infant. Based upon observations made primarily in adult rodents and the fact that the immature brain uses proteolytic systems for cell migration and growth, we hypothesized that thrombin and plasmin enzyme activities contribute to the brain damage after PVH. The viability of mixed brain cells derived from newborn rat periventricular region was suppressed by whole blood and thrombin, but not plasmin. Following injection of autologous blood into the periventricular region of newborn rat brain, proteolytic activity was detected in a halo around the hematoma using membrane overlays impregnated with thrombin and plasmin fluorogenic substrates. Two-day old rats received periventricular injection of blood, thrombin, and plasminogen. After 2 days, thrombin and blood were associated with significantly greater damage than saline or plasminogen. Two-day old mice received intracerebral injections of blood in combination with saline or the proteolytic inhibitors hirudin, alpha2macroglobulin, or plasminogen activator inhibitor-1. After 2 days, hirudin significantly reduced brain cell death and inflammation. Two-day-old mice then received low and high doses of hirudin mixed with blood after which behavioral testing was conducted repeatedly. At 10 weeks there was no statistically significant evidence for behavioral or structural brain protection. These results indicate that thrombin likely plays a role in neonatal periventricular brain damage following PVH. However, additional factors are likely important in the recovery from this result.

Periventricular/intraventricular hemorrhage in neonatal mouse cerebrum.

Periventricular/intraventricular hemorrhage (PVH/IVH) into brain can occur in premature infants and is associated with poor developmental outcome. The purpose of this study was to develop and characterize a model of PVH/IVH in newborn mouse. We hypothesized that periventricular germinal matrix would exhibit reduced cell proliferation. PVH/IVH was induced in 1-day-old mice by injection of autologous blood into the periventricular tissue. Magnetic resonance images (MRI) were obtained from 15 minutes to 14 days later. Mice were killed 4 hours to 28 days later. Cell proliferation, dying cells, astrocyte and microglial reactions, neutrophils, and lymphocytes were quantified. Histological studies showed that MRI accurately localizes the hematoma but overestimates its size. The hematoma, located in the striatum and germinal tissue, always extended into the lateral ventricles. Cell proliferation, measured by Ki67 immunoreactivity, was suppressed bilaterally in germinal matrix and beyond from 8 hours to 7 days. Increased cell death was observed in the ipsilateral striatum and germinal matrix 1 and 2 days after PVH/IVH. Astrocyte and microglia reaction peaked at 2 days and persisted up to 28 days. Inflammatory response was minimal. Extravasated blood might play an important role in brain damage following PVH/IVH through suppression of cell proliferation.

Active inflammation increases the heterogeneity of MRI texture in mice with relapsing experimental allergic encephalomyelitis.

Inflammation modulates tissue damage in relapsing-remitting multiple sclerosis (MS) both acutely and chronically, but its severity is difficult to evaluate with conventional MRI analysis. In mice with experimental allergic encephalomyelitis (EAE, a model of MS), we administered ultra small particles of iron oxide to track macrophage-mediated inflammation during the onset (relapse) and recovery (remission) of disease activity using high field MRI. We performed MRI texture analysis, a sensitive measure of tissue regularity, and T2 assessment both in EAE lesions and the control tissue, and measured spinal cord volume. We found that inflammation was 3 times more remarkable at onset than at recovery of EAE in histology yet demyelination appeared similar across animals and disease course. In MRI, lesion texture was more heterogeneous; T2 was lower; and spinal cord volume was greater in EAE than in controls, but only MRI texture was worse at relapse than at remission of EAE. Moreover, MRI texture correlated with spinal cord volume and tended to correlate with the extent of disability in EAE. While subject to further confirmation, our findings may suggest the sensitivity of MRI texture analysis for accessing inflammation.

A cognitive rehabilitation paradigm effective in male rats lacks efficacy in female rats.

Cognitive dysfunction, as a consequence of dementia, is a significant cause of morbidity lacking efficacious treatment. Females comprise at least half of this demographic but have been vastly underrepresented in preclinical studies. The current study addressed this gap by assessing the protective efficacy of physical exercise and cognitive activity on learning and memory outcomes in a rat model of vascular dementia. Forty ovariectomized Sprague-Dawley rats (∼6 months old) were exposed to either a diet high in saturated fats and refined sugars or standard laboratory chow and underwent either chronic bilateral carotid occlusion or Sham surgery. Learning and memory abilities were evaluated using standard cognitive outcomes over the ensuing 6 months, followed by histologic analyses of hippocampal CA1 neurons. In Experiment 1, we confirmed hypoperfusion-induced cognitive dysfunction using a 2 × 2 (Surgery × Diet) experimental design, without alterations in hippocampal architecture. In Experiment 2, hypoperfused animals were either exposed to alternating days of physical (wheel running) and cognitive activity (modified Hebb-Williams maze) or sedentary housing. In contrast to males, this combination rehabilitation paradigm did not improve cognition or histopathologic outcomes in hypoperfused animals. These findings, highlighting differences between female and male animals, show the necessity of including both sexes in preclinical experimentation.

Cognitive rehabilitation reduces cognitive impairment and normalizes hippocampal CA1 architecture in a rat model of vascular dementia.

Dementia is a major cause of morbidity in the western society. Pharmacological therapies to delay the progression of cognitive impairments are modestly successful. Consequently, new therapies are urgently required to improve cognitive deficits associated with dementia. We evaluated the effects of physical and cognitive activity on learning and memory in a rat model of vascular dementia (VasD). Male Sprague-Dawley rats (6 months old) were exposed to either regular chow or a diet rich in saturated fats and sucrose and chronic bilateral common carotid artery occlusion or sham surgery. First, this model of VasD was validated using a 2 × 2 experimental design (surgery × diet) and standard cognitive outcomes. Next, using identical surgical procedures, we exposed animals to a paradigm of cognitive rehabilitation or a sedentary condition. At 16 weeks post surgery, VasD animals demonstrated significant learning and memory deficits in the Morris water maze, independent of diet. Rehabilitation significantly attenuated these cognitive deficits at this time point as well as at 24 weeks. Further, rehabilitation normalized hippocampal CA1 soma size (area and volume) to that of control animals, independent of cell number. Importantly, these findings demonstrate beneficial neuroplasticity in early middle-aged rats that promoted cognitive recovery, an area rarely explored in preclinical studies.

Blood-brain barrier disruption in CCL2 transgenic mice during pertussis toxin-induced brain inflammation.

BACKGROUND: The chemokine CCL2 has an important role in the recruitment of inflammatory cells into the central nervous system (CNS). A transgenic mouse model that overexpresses CCL2 in the CNS shows an accumulation of leukocytes within the perivascular space surrounding vessels, and which infiltrate into the brain parenchyma following the administration of pertussis toxin (PTx). METHODS: This study used contrast-enhanced magnetic resonance imaging (MRI) to quantify the extent of blood-brain barrier (BBB) disruption in this model pre- and post-PTx administration compared to wild-type mice. Contrast-enhanced MR images were obtained before and 1, 3, and 5 days after PTx injection in each animal. After the final imaging session fluorescent dextran tracers were administered intravenously to each mouse and brains were examined histologically for cellular infiltrates, BBB leakage and tight junction protein. RESULTS: BBB breakdown, defined as a disruption of both the endothelium and glia limitans, was found only in CCL2 transgenic mice following PTx administration and seen on MR images as focal areas of contrast enhancement and histologically as dextrans leaking from blood vessels. No evidence of disruption in endothelial tight junctions was observed. CONCLUSION: Genetic and environmental stimuli were needed to disrupt the integrity of the BBB in this model of neuroinflammation.

Magnetic resonance imaging of blood-spinal cord barrier disruption in mice with experimental autoimmune encephalomyelitis.

Inflammation, demyelination, and blood-spinal cord barrier (BSB) breakdown occur in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. The purpose of this study was to evaluate the utility of MRI for detecting lesions and BSB disruption in vivo during EAE in the mouse lumbar spinal cord, to determine how MR features of BSB disruption change during the course of disease, and to relate such changes to clinical signs and histological features of disease. Following induction of EAE in C57BL/6 mice, contrast-enhanced (CE) T(1)-weighted MR images were acquired to detect BSB disruption in the lumbar spinal cord at the early stage of disease, at peak disease, and at remission, and T(2)-weighted images were obtained to monitor spinal cord morphology. Following imaging the spinal cords were assessed in situ for general features of inflammation, BSB leakage, activated macrophages/microglia, and demyelination. No focal lesions were evident on T(2)-weighted MR images. BSB disruption was greatest at the onset of signs of disease, and decreased progressively thereafter. Inflammation and demyelination were pronounced at the initial stage of disease and at peak disease, and were decreased at remission. Nonuniform contrast enhancement indicated that breakdown of the BSB occurred predominantly within the white matter (WM) of the spinal cord.

West Nile virus-induced neuroinflammation: glial infection and capsid protein-mediated neurovirulence.

West Nile virus (WNV) infection causes neurological disease at all levels of the neural axis, accompanied by neuroinflammation and neuronal loss, although the underlying mechanisms remain uncertain. Given the substantial activation of neuroinflammatory pathways observed in WNV infection, we hypothesized that WNV-mediated neuroinflammation and cell death occurred through WNV infection of both glia and neurons, which was driven in part by WNV capsid protein expression. Analysis of autopsied neural tissues from humans with WNV encephalomyelitis (WNVE) revealed WNV infection of both neurons and glia. Upregulation of proinflammatory genes, CXCL10, interleukin-1beta, and indolamine-2',3'-deoxygenase with concurrent suppression of the protective astrocyte-specific endoplasmic reticulum stress sensor gene, OASIS (for old astrocyte specifically induced substance), was evident in WNVE patients compared to non-WNVE controls. These findings were supported by increased ex vivo expression of these proinflammatory genes in glia infected by WNV-NY99. WNV infection caused endoplasmic reticulum stress gene induction and apoptosis in neurons but did not affect glial viability. WNV-infected astrocytic cells secreted cytotoxic factors, which caused neuronal apoptosis. The expression of the WNV-NY99 capsid protein in neurons and glia by a Sindbis virus-derived vector (SINrep5-WNVc) caused neuronal death and the release of neurotoxic factors by infected astrocytes, coupled with proinflammatory gene induction and suppression of OASIS. Striatal implantation of SINrep5-WNV(C) induced neuroinflammation in rats, together with the induction of CXCL10 and diminished OASIS expression, compared to controls. Moreover, magnetic resonance neuroimaging showed edema and tissue injury in the vicinity of the SINrep5-WNVc implantation site compared to controls, which was complemented by neurobehavioral abnormalities in the SINrep5-WNVc-implanted animals. These studies underscore the important interactions between the WNV capsid protein and neuroinflammation in the pathogenesis of WNV-induced neurological disorders.

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.

Metalloproteinases control brain inflammation induced by pertussis toxin in mice overexpressing the chemokine CCL2 in the central nervous system.

Inflammatory leukocytes infiltrate the CNS parenchyma in neuroinflammation. This involves cellular migration across various structures associated with the blood-brain barrier: the vascular endothelium, the glia limitans, and the perivascular space between them. Leukocytes accumulate spontaneously in the perivascular space in brains of transgenic (Tg) mice that overexpress CCL2 under control of a CNS-specific promoter. The Tg mice show no clinical symptoms, even though leukocytes have crossed the endothelial basement membrane. Pertussis toxin (PTx) given i.p. induced encephalopathy and weight loss in Tg mice. We used flow cytometry, ultra-small superparamagnetic iron oxide-enhanced magnetic resonance imaging, and immunofluorescent staining to show that encephalopathy involved leukocyte migration across the glia limitans into the brain parenchyma, identifying this as the critical step in inducing clinical symptoms. Metalloproteinase (MPs) enzymes are implicated in leukocyte infiltration in neuroinflammation. Unmanipulated Tg mice had elevated expression of tissue inhibitor of metalloproteinase-1, matrix metalloproteinase (MMP)-10, and -12 mRNA in the brain. PTx further induced expression of tissue inhibitor of metalloproteinase-1, metalloproteinase disintegrins-12, MMP-8, and -10 in brains of Tg mice. Levels of the microglial-associated MP MMP-15 were not affected in control or PTx-treated Tg mice. PTx also up-regulated expression of proinflammatory cytokines IL-1beta and TNF-alpha mRNA in Tg CNS. Weight loss and parenchymal infiltration, but not perivascular accumulation, were significantly inhibited by the broad-spectrum MP inhibitor BB-94/Batimastat. Our finding that MPs mediate PTx-induced parenchymal infiltration to the chemokine-overexpressing CNS has relevance for the pathogenesis of human diseases involving CNS inflammation, such as multiple sclerosis.

A novel MRI texture analysis of demyelination and inflammation in relapsing-remitting experimental allergic encephalomyelitis.

We have developed a novel multiscale localized image texture analysis technique, based upon the polar Stockwell Transform (PST). In this paper we characterized image texture in vivo using the PST in histologically verified lesion areas in T2-weighted MRI of an animal model of multiple sclerosis. Both high and low frequency signals, representing inflammation and demyelination, were significantly increased in pathological regions compared to normal control tissue. This suggests that this new local spatial-frequency measure of image texture may provide a sensitive and precise indication of disease activity.

Dietary supplementation of omega-3 polyunsaturated fatty acids worsens forelimb motor function after intracerebral hemorrhage in rats.

Dietary intake of omega-3 polyunsaturated fatty acids has been associated with decreased clotting ability and increased risk of hemorrhagic stroke. The aim of the current study was to assess the effect of dietary supplementation of omega-3 polyunsaturated fatty acid on functional outcome after hemorrhagic stroke. Rats were maintained on a diet containing approximately 30% of energy as either fish oil (rich in omega-3 fatty acids) or safflower oil (rich in omega-6 fatty acids) and subjected to either intracerebral hemorrhage or sham surgery. Behavioral tests, infarct measurement, and MR imaging techniques were used to assess outcome. While there was no significant difference in infarct volume between rats on different diets, animals maintained on a diet enriched with fish oil exhibited increased cerebral blood flow after surgery. These animals were significantly more impaired than rats fed the safflower-oil-enriched diet in tests of forelimb dexterity and fine motor control. These results suggest that high intake of omega-3 polyunsaturated fatty acids may not only increase the risk of hemorrhagic stroke as shown in previous studies, but most importantly may lead to a more severe motor impairment and a poorer functional outcome after such an event.

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.