Clinical Features, Non-Contrast CT Radiomic and Radiological Signs in Models for the Prediction of Hematoma Expansion in Intracerebral Hemorrhage.

PURPOSE: Rapid identification of hematoma expansion (HE) risk at baseline is a priority in intracerebral hemorrhage (ICH) patients and may impact clinical decision making. Predictive scores using clinical features and Non-Contract Computed Tomography (NCCT)-based features exist, however, the extent to which each feature set contributes to identification is limited. This paper aims to investigate the relative value of clinical, radiological, and radiomics features in HE prediction. METHODS: Original data was retrospectively obtained from three major prospective clinical trials ["Spot Sign" Selection of Intracerebral Hemorrhage to Guide Hemostatic Therapy (SPOTLIGHT)NCT01359202; The Spot Sign for Predicting and Treating ICH Growth Study (STOP-IT)NCT00810888] Patients baseline and follow-up scans following ICH were included. Clinical, NCCT radiological, and radiomics features were extracted, and multivariate modeling was conducted on each feature set. RESULTS: 317 patients from 38 sites met inclusion criteria. Warfarin use (p=0.001) and GCS score (p=0.046) were significant clinical predictors of HE. The best performing model for HE prediction included clinical, radiological, and radiomic features with an area under the curve (AUC) of 87.7%. NCCT radiological features improved upon clinical benchmark model AUC by 6.5% and a clinical & radiomic combination model by 6.4%. Addition of radiomics features improved goodness of fit of both clinical (p=0.012) and clinical & NCCT radiological (p=0.007) models, with marginal improvements on AUC. Inclusion of NCCT radiological signs was best for ruling out HE whereas the radiomic features were best for ruling in HE. CONCLUSION: NCCT-based radiological and radiomics features can improve HE prediction when added to clinical features.

A Guide to Extract Spinal Cord for Translational Stem Cell Biology Research: Comparative Analysis of Adult Human, Porcine, and Rodent Spinal Cord Stem Cells.

Improving the clinical translation of animal-based neural stem/progenitor cell (NSPC) therapies to humans requires an understanding of intrinsic human and animal cell characteristics. We report a novel in vitro method to assess spinal cord NSPCs from a small (rodent) and large (porcine) animal model in comparison to human NSPCs. To extract live adult human, porcine, and rodent spinal cord tissue, we illustrate a strategy using an anterior or posterior approach that was simulated in a porcine model. The initial expansion of primary NSPCs is carried out using the neurosphere assay followed by a pharmacological treatment phase during which NSPCs derived from humans, porcines, and rodents are assessed in parallel using the same defined parameters. Using this model, NSPCs from all species demonstrated multi-lineage differentiation and self-renewal. Importantly, these methods provide conditions to enable the direct comparison of species-dependent cell behavior in response to specific exogenous signals.

The Impact of Covert Lacunar Infarcts and White Matter Hyperintensities on Cognitive and Motor Outcomes After Stroke.

BACKGROUND AND AIMS: In addition to overt stroke lesions, co-occurring covert lesions, including white matter hyperintensities (WMH) and covert lacunar infarcts (CLI), contribute to poststroke outcome. The purpose of this study was to examine the relationship between covert lesions, and motor and cognitive outcomes in individuals with chronic stroke. METHODS: Volumetric quantification of clinically overt strokes, covert lesions (periventricular and deep: pWMH, dWMH, pCLI, dCLI), ventricular and sulcal CSF (vCSF, sCSF), and normal appearing white (NAWM) and gray matter (NAGM) was performed using structural magnetic resonance imaging. We assessed motor impairment and function, and global cognition, memory, and other cognitive domains. When correlation analysis identified more than one MR parameter relating to stroke outcomes, we used regression modeling to identify which factor had the strongest impact. RESULTS: Neuropsychological and brain imaging data were collected from 30 participants at least 6 months following a clinically diagnosed stroke. Memory performance related to vCSF (r = -0.52, P = .004). The strongest predictor of nonmemory domains was pCLI (r2 = 0.28, P = .004). Motor impairment and function were most strongly predicted by the volume of stroke and NAWM (r2 = 0.36; P = .001), and dWMH (r2 = 0.39; P = .001) respectively. CONCLUSIONS: Covert lesion type and location have important consequences for post-stroke cognitive and motor outcome. Limiting the progression of covert lesions in aging populations may enhance the degree of recovery post-stroke.

A structural motor network correlates with motor function and not impairment post stroke.

Combining structural and functional magnetic resonance imaging may provide insight into how residual motor networks contribute to motor outcomes post-stroke. The purpose of this study was to examine whether a structural motor network (SMN), generated with fMRI guided diffusion-based tractography, relates to motor function post-stroke. Twenty-seven individuals with mild to moderate upper limb impairment post stroke underwent diffusion magnetic resonance imaging. A bilateral motor network mask guided white matter tractography for each participant. Fractional anisotrophy (FA) was calculated for the SMN and corticospinal tracts (CST). The Wolf Motor Function Test (WMFT) rate and Fugl-Meyer Upper Limb (FM) tests characterized arm function and impairment respectively. The SMN and ipsilesional CST together explained approximately 35% of the variance in paretic arm function (WMFT-rate p=0.006). This study demonstrates that a broader motor network, like the SMN, is functionally meaningful. Given that the motor network is widely distributed, the proposed SMN warrants further investigation as a potential adjunct biomarker to characterize recovery potential after stroke.

A reliability assessment of constrained spherical deconvolution-based diffusion-weighted magnetic resonance imaging in individuals with chronic stroke.

BACKGROUND: Diffusion-weighted magnetic resonance imaging (DW-MRI) is commonly used to assess white matter properties after stroke. Novel work is utilizing constrained spherical deconvolution (CSD) to estimate complex intra-voxel fiber architecture unaccounted for with tensor-based fiber tractography. However, the reliability of CSD-based tractography has not been established in people with chronic stroke. NEW METHOD: Establishing the reliability of CSD-based DW-MRI in chronic stroke. High-resolution DW-MRI was performed in ten adults with chronic stroke during two separate sessions. Deterministic region of interest-based fiber tractography using CSD was performed by two raters. Mean fractional anisotropy (FA), apparent diffusion coefficient (ADC), tract number, and tract volume were extracted from reconstructed fiber pathways in the corticospinal tract (CST) and superior longitudinal fasciculus (SLF). Callosal fiber pathways connecting the primary motor cortices were also evaluated. Inter-rater and test-retest reliability were determined by intra-class correlation coefficients (ICCs). RESULTS: ICCs revealed excellent reliability for FA and ADC in ipsilesional (0.86-1.00; p<0.05) and contralesional hemispheres (0.94-1.00; p<0.0001), for CST and SLF fibers; and excellent reliability for all metrics in callosal fibers (0.85-1.00; p<0.05). ICC ranged from poor to excellent for tract number and tract volume in ipsilesional (-0.11 to 0.92; p≤0.57) and contralesional hemispheres (-0.27 to 0.93; p≤0.64), for CST and SLF fibers. COMPARISON WITH EXISTING METHOD: Like other select DW-MRI approaches, CSD-based tractography is a reliable approach to evaluate FA and ADC in major white matter pathways, in chronic stroke. CONCLUSION: Future work should address the reproducibility and utility of CSD-based metrics of tract number and tract volume.

A Review of Transcranial Magnetic Stimulation and Multimodal Neuroimaging to Characterize Post-Stroke Neuroplasticity.

Following stroke, the brain undergoes various stages of recovery where the central nervous system can reorganize neural circuitry (neuroplasticity) both spontaneously and with the aid of behavioral rehabilitation and non-invasive brain stimulation. Multiple neuroimaging techniques can characterize common structural and functional stroke-related deficits, and importantly, help predict recovery of function. Diffusion tensor imaging (DTI) typically reveals increased overall diffusivity throughout the brain following stroke, and is capable of indexing the extent of white matter damage. Magnetic resonance spectroscopy (MRS) provides an index of metabolic changes in surviving neural tissue after stroke, serving as a marker of brain function. The neural correlates of altered brain activity after stroke have been demonstrated by abnormal activation of sensorimotor cortices during task performance, and at rest, using functional magnetic resonance imaging (fMRI). Electroencephalography (EEG) has been used to characterize motor dysfunction in terms of increased cortical amplitude in the sensorimotor regions when performing upper limb movement, indicating abnormally increased cognitive effort and planning in individuals with stroke. Transcranial magnetic stimulation (TMS) work reveals changes in ipsilesional and contralesional cortical excitability in the sensorimotor cortices. The severity of motor deficits indexed using TMS has been linked to the magnitude of activity imbalance between the sensorimotor cortices. In this paper, we will provide a narrative review of data from studies utilizing DTI, MRS, fMRI, EEG, and brain stimulation techniques focusing on TMS and its combination with uni- and multimodal neuroimaging methods to assess recovery after stroke. Approaches that delineate the best measures with which to predict or positively alter outcomes will be highlighted.

Compensatory motor network connectivity is associated with motor sequence learning after subcortical stroke.

Following stroke, functional networks reorganize and the brain demonstrates widespread alterations in cortical activity. Implicit motor learning is preserved after stroke. However the manner in which brain reorganization occurs, and how it supports behavior within the damaged brain remains unclear. In this functional magnetic resonance imaging (fMRI) study, we evaluated whole brain patterns of functional connectivity during the performance of an implicit tracking task at baseline and retention, following 5 days of practice. Following motor practice, a significant difference in connectivity within a motor network, consisting of bihemispheric activation of the sensory and motor cortices, parietal lobules, cerebellar and occipital lobules, was observed at retention. Healthy subjects demonstrated greater activity within this motor network during sequence learning compared to random practice. The stroke group did not show the same level of functional network integration, presumably due to the heterogeneity of functional reorganization following stroke. In a secondary analysis, a binary mask of the functional network activated from the aforementioned whole brain analyses was created to assess within-network connectivity, decreasing the spatial distribution and large variability of activation that exists within the lesioned brain. The stroke group demonstrated reduced clusters of connectivity within the masked brain regions as compared to the whole brain approach. Connectivity within this smaller motor network correlated with repeated sequence performance on the retention test. Increased functional integration within the motor network may be an important neurophysiological predictor of motor learning-related change in individuals with stroke.

Prolonged therapeutic hypothermia does not adversely impact neuroplasticity after global ischemia in rats.

Hypothermia improves clinical outcome after cardiac arrest in adults. Animal data show that a day or more of cooling optimally reduces edema and tissue injury after cerebral ischemia, especially after longer intervention delays. Lengthy treatments, however, may inhibit repair processes (e.g., synaptogenesis). Thus, we evaluated whether unilateral brain hypothermia (∼33°C) affects neuroplasticity in the rat 2-vessel occlusion model. In the first experiment, we cooled starting 1 hour after ischemia for 2, 4, or 7 days. Another group was cooled for 2 days starting 48 hours after ischemia. One group remained normothermic throughout. All hypothermia treatments started 1 hour after ischemia equally reduced hippocampal CA1 injury in the cooled hemisphere compared with the normothermic side and the normothermic group. Cooling only on days 3 and 4 was not beneficial. Importantly, no treatment influenced neurogenesis (Ki67/Doublecortin (DCX) staining), synapse formation (synaptophysin), or brain-derived neurotropic factor (BDNF) immunohistochemistry. A second experiment confirmed that BDNF levels (ELISA) were equivalent in normothermic and 7-day cooled rats. Last, we measured zinc (Zn), which is important in plasticity, with X-ray fluorescence imaging in normothermic and 7-day cooled rats. Hypothermia did not alter the postischemic distribution of Zn within the hippocampus. In summary, cooling significantly mitigates injury without compromising neuroplasticity.

Ferric iron chelation lowers brain iron levels after intracerebral hemorrhage in rats but does not improve outcome.

Iron-mediated free radical damage contributes to secondary damage after intracerebral hemorrhage (ICH). Iron is released from heme after hemoglobin breakdown and accumulates in the parenchyma over days and then persists in the brain for months (e.g., hemosiderin). This non-heme iron has been linked to cerebral edema and cell death. Deferoxamine, a ferric iron chelator, has been shown to mitigate iron-mediated damage, but results vary with less protection in the collagenase model of ICH. This study used rapid-scanning X-ray fluorescence (RS-XRF), a synchrotron-based imaging technique, to spatially map total iron and other elements (zinc, calcium and sulfur) at three survival times after collagenase-induced ICH in rats. Total iron was compared to levels of non-heme iron determined by a Ferrozine-based spectrophotometry assay in separate animals. Finally, using RS-XRF we measured iron levels in ICH rats treated with deferoxamine versus saline. The non-heme iron assay showed elevations in injured striatum at 3 days and 4 weeks post-ICH, but not at 1 day. RS-XRF also detected significantly increased iron levels at comparable times, especially notable in the peri-hematoma zone. Changes in other elements were observed in some animals, but these were inconsistent among animals. Deferoxamine diminished total parenchymal iron levels but did not attenuate neurological deficits or lesion volume at 7 days. In summary, ICH significantly increased non-heme and total iron levels. We evaluated the latter and found it to be significantly lowered by deferoxamine, but its failure to attenuate injury or functional impairment in this model raises concern about successful translation to patients.

Prolonged hypothermia in rat: a safety study using brain-selective and systemic treatments.

Hypothermia is an effective neuroprotectant for cardiac arrest and perinatal ischemic injury. Hypothermia also improves outcome after traumatic brain injury and stroke. Although the ideal treatment parameters (duration, delay, and depth) are not fully delineated, prolonged cooling is usually more effective than shorter periods. There is the concern that extended cooling may be hazardous to brain plasticity and cause damage. In order to evaluate this possibility, we assessed the effects of 3 days of systemic hypothermia (32°C) in rats subjected to a sham stroke surgery. There were no detrimental behavioral effects or signs of brain damage. As even longer cooling may be needed in some patients, we cooled (∼32°C) the right hemisphere of rats for 3 or 21 days. Physiological variables, functional outcome, and measures of cell injury were examined. Focal brain cooling for 21 days modestly decreased heart rate, blood pressure, and core temperature. However, focal hypothermia did not affect subsequent behavior (e.g., spontaneous limb usage), cell morphology (e.g., dendritic arborization, ultrastructure), or cause cell death. In conclusion, prolonged mild hypothermia did not harm the brain of normal animals. Further research is now needed to evaluate whether such treatments affect plasticity after brain injury.

Rehabilitation promotes recovery after whole blood-induced intracerebral hemorrhage in rats.

BACKGROUND: Rehabilitation improves recovery after intracerebral hemorrhage (ICH) caused by collagenase infusion into the striatum of rats by promoting dendritic growth and reducing brain injury in this model. OBJECTIVE: Effective preclinical testing requires multiple models because none, including the collagenase model, perfectly mimics human ICH. Thus, the authors assessed enhanced rehabilitation (ER), a combination of environmental enrichment and task-specific motor training, on skilled reaching, lesion size, and dendritic plasticity after whole blood-induced, striatal ICH. METHODS: Three groups of rats were trained to retrieve food in a reaching task prior to ICH. One group was euthanized at 7 days, whereas 2 groups survived 7 weeks post-ICH. Of the latter, 1 group received 2 weeks of ER starting at 7 days, whereas controls did not. Reaching success was assessed 6 weeks after ICH. Lesion volume and dendritic length and complexity (contralateral striatum) were assessed. RESULTS: The ICH caused reaching deficits that were markedly attenuated by ER as observed previously in the collagenase model. In contrast to that model, there was a time-dependent decline in dendritic length after untreated, whole blood-induced ICH. Furthermore, behavioral recovery was not accompanied by changes in lesion volume or contralateral dendritic morphology. CONCLUSIONS: Converging data from animal models support the use of rehabilitation for ICH patients. However, although rehabilitation effectively promotes behavioral recovery, the mechanisms of action vary by model making it difficult to predict clinical effects.

Intravascular stem cell transplantation for stroke.

Stroke is the third leading cause of death and the leading cause of adult disability in North America. Emphasis has been placed on developing treatments that reduce the devastating long-term impacts of this disease, and preclinical research on stem cell therapy has demonstrated promising results. However, questions about the optimal cell delivery method and timing of cell transplantation are not fully answered. Recent findings suggest that intravascular stem cell delivery is a safe and efficacious alternative to stereotactic cell injections. It also offers advantages should repeat treatments prove beneficial. Recent reports further suggest that intra-arterial injection results in a wider distribution of cells throughout the stroked hemisphere with a significantly greater cell engraftment compared to intravenous injection. In this review, we describe the benefits and potential risks associated with intravascular stem cell delivery and compare intra-arterial to intravenous cell transplantation methods. We discuss the importance of cell biodistribution and timing of transplantation in driving cell survival. We examine current proposed mechanisms involved in cell migration and functional recovery and discuss future directions for intravascular stem cell therapy research.

Rodent models of intracerebral hemorrhage.

The collagenase and whole blood intracerebral hemorrhage (ICH) models are widely used to identify mechanisms of injury and to evaluate treatments. Despite preclinical successes, to date, no treatment tested in phase III clinical trials has benefited ICH patients. These failures call into question the predictive value of current ICH models. By highlighting differences between these common rodent models of ICH, we sought to help investigators choose the more appropriate model for their study and to encourage the use of both whenever possible. For instance, we previously reported substantial differences in the bleeding profile, progression of cell death, and functional outcome between these models. These and other differences influence the efficacy and mechanisms of action of various treatment modalities. Thus, in this review, we also summarize neuroprotective and rehabilitation findings in each model. We conclude that differences between ICH models along with our current inability to identify the more clinically predictive model necessitate that preclinical assessments should normally be done in both. Such an approach, coupled with better assessment practices, will likely improve chances of future clinical success.

Rehabilitation after intracerebral hemorrhage in rats improves recovery with enhanced dendritic complexity but no effect on cell proliferation.

Rehabilitation, consisting of enriched environment and skilled reach training, improves recovery after intracerebral hemorrhage (ICH) in rats. We tested whether rehabilitation influences dendritic morphology (Golgi-Cox staining-experiment 1) or cell proliferation (immunohistochemistry-experiment 2). In the latter experiment, BrdU was given from 14 to 18 days post stroke, and cells were labeled for BrdU along with NeuN, Iba1 or GFAP. One week after a striatal ICH, via collagenase infusion, the rats were given rehabilitation for 2 weeks or control treatment (group housing in standard cages). Behavioral outcome (e.g., skilled reaching, walking) was assessed at multiple times. Rats were euthanized at 5 (experiment 2) or 6 (experiment 1) weeks post-ICH. As expected, rehabilitation significantly improved skilled reaching and walking ability. There was also a concomitant increase in dendritic length in peri-hematoma striatum and ipsilateral cortex as well as in the contralateral striatum. Lesion volume did not differ between groups, nor did cell proliferation. There was no evidence of neurogenesis, but there was increased Iba1 and GFAP labeling in the injured hemisphere. Thus, rehabilitation likely improves outcome after ICH though a plasticity response (e.g., increased dendritic growth) that does not involve neurogenesis.

Failure of deferoxamine, an iron chelator, to improve outcome after collagenase-induced intracerebral hemorrhage in rats.

Intracerebral hemorrhage (ICH) is a devastating stroke with no clinically proven treatment. Deferoxamine (DFX), an iron chelator, is a promising therapy that lessens edema, mitigates peri-hematoma cell death, and improves behavioral recovery after whole-blood-induced ICH in rodents. In this model, blood is directly injected into the brain, usually into the striatum. This mimics many but not all clinical features of ICH (e.g., there is no spontaneous bleed). Thus, we tested whether DFX improves outcome after collagenase-induced striatal ICH in rats. In the first experiment, 3- and 7-day DFX regimens (100 mg/kg twice per day starting 6 h after ICH), similar to those shown effective in the whole-blood model, were compared to saline treatment. Functional recovery was evaluated from 3 to 28 days with several behavioral tests. Except for one instance, DFX failed to lessen ICH-induced behavioral impairments and it did not lessen brain injury, which averaged 43.5 mm(3) at a 28-day survival. In the second experiment, 3 days of DFX treatment were given starting 0 or 6 h after collagenase infusion. Striatal edema occurred, but it was not affected by either DFX treatment (vs. saline treatment). Therefore, in contrast to studies using the whole-blood model, DFX treatment did not improve outcome in the collagenase model. Our findings, when compared to others, suggest that there are critical differences between these ICH models. Perhaps, the current clinical work with DFX will help identify the more clinically predictive model for future neuroprotection studies.

Delayed rehabilitation lessens brain injury and improves recovery after intracerebral hemorrhage in rats.

Rehabilitation improves recovery after intracerebral hemorrhage (ICH) in rats. In some cases, brain damage is attenuated. In this study, we tested whether environmental enrichment (EE) combined with skilled reach training improves recovery and lessens brain injury after ICH in rats. Collagenase was injected stereotaxically to produce a moderate-sized striatal ICH. One week after ICH rats were either placed into a rehabilitation (REHAB) or control (CONT) condition. The REHAB rats received 15 h of EE and four 15-minute reach-training sessions daily over 5 days a week for 2 weeks. The CONT rats stayed in standard group cages. Skilled reaching (staircase test), walking (horizontal ladder) and forelimb use bias (cylinder test) were assessed at 4 and 6 weeks after ICH. Lesion volume, corpus callosum volume and cortical thickness were calculated 46 days after ICH. The REHAB treatment reduced lesion volume by 28% (p=0.019) without affecting the corpus callosum volume (p=0.405) or cortical thickness (p=0.300), thus indicating that protection was due to lessening striatal injury. As well, REHAB significantly improved skilled reaching ability in the staircase apparatus at 4 (p=0.002) and 6 weeks (p<0.001) post-ICH. Transient benefit was obtained in the ladder test at 4 weeks (p=0.021). Unexpectedly, REHAB treatment lessened spontaneous use of the contralateral-to-ICH limb at 4 (p=0.045) and 6 weeks (p=0.041). In summary, the combination of EE and reach training significantly attenuates lesion volume (striatal injury) while improving skilled reaching and walking ability. These findings encourage the use of early rehabilitation therapies in patients suffering from basal ganglia hemorrhaging.

Influence of amphetamine on recovery after intracerebral hemorrhage in rats.

D-amphetamine (AMP) paired with physical activity (e.g., beam walking) improves recovery after ischemic injury to the cortical motor system of rodents. We tested whether AMP promotes recovery after intracerebral hemorrhage (ICH) in rats. A moderate-sized ICH was produced by stereotaxically injecting collagenase into the striatum. Five days later rats were placed into either environmental enrichment cages (EE) or a control condition (group housing in standard cages) until euthanasia at 4 weeks post-ICH. Animals were injected with either AMP (2 mg/kg i.p.) or sterile saline on days 7, 9 and 11 after ICH. Rats in EE also received training on beam (walking) and tray (skilled reaching) tasks 30 min after each injection. Walking (beam and ladder task), skilled reaching (tray and staircase tasks) and neurological deficits (NDS) were repeatedly assessed. We predicted that EE would improve recovery and that AMP would further enhance it. Results showed that EE, but not AMP, significantly and consistently improved recovery on the beam and ladder task. Neither treatment significantly affected skilled reaching. Lesion volume was not significantly different among groups (overall average: 44.6 mm(3) of tissue lost +/-15.3 S.D.). In conclusion, EE provides modest benefit for striatal ICH whereas AMP does not. This suggests that AMP will not provide substantial benefit to those patients with severe ICH affecting the basal ganglia.

Forced exercise does not improve recovery after hemorrhagic stroke in rats.

Exercise can improve recovery following ischemia and intracerebral hemorrhage (ICH) in rodents. We tested whether forced exercise (EX; running wheel) prior to and/or following ICH in rats would reduce lesion volume and improve functional outcome (walking, skilled reaching, spontaneous paw usage) at 7 weeks post-ICH. A striatal hemorrhage was produced by infusing collagenase. First, we compared animals that received EX (2 weeks; 1 h/day) ending two days prior to ICH and/or starting two weeks following ICH. EX did not improve functional recovery or affect lesion size. Doubling the amount of EX given per day (two 1-h sessions) both prior to and following ICH did not alter lesion volume, but worsened recovery. We then determined if EX (1 h/day) prior to and following ICH would affect outcome after a somewhat milder insult. There were no differences between the groups in lesion volume or recovery. Finally, we used a hemoglobin assay at 12 h following ICH to determine if pre-stroke EX (2 weeks; 1 h/day) aggravated bleeding. It did not. These observations suggest that EX does not improve outcome when given prior to and/or when delayed following ICH. Effective rehabilitation for ICH will likely require more complex interventions than forced running.

Gauging recovery after hemorrhagic stroke in rats: implications for cytoprotection studies.

Successful clinical translation of prospective cytoprotectants will likely occur only with treatments that improve functional recovery in preclinical (rodent) studies. Despite this assumption, many rely solely on histopathologic end points or the use of one or two simple behavioral tests. Presently, we used a battery of tests to gauge recovery after a unilateral intracerebral hemorrhagic stroke (ICH) targeting the striatum. In total, 60 rats (N=15 per group) were stereotaxically infused with 0 (SHAM), 0.06 (MILD lesion), 0.12 (MODERATE lesion), or 0.18 U (SEVERE lesion) of bacterial collagenase. This created a range of injury akin to moderate (from SEVERE to MODERATE or MODERATE to MILD lesion size approximately 30% reduction) and substantial cytoprotection (SEVERE to MILD lesion size--51% reduction). Post-ICH functional testing occurred over 30 days. Tests included the horizontal ladder and elevated beam tests, swimming, limb-use asymmetry (cylinder) test, a Neurologic Deficit Scale, an adhesive tape removal test of sensory neglect, and the staircase and single pellet tests of skilled reaching. Most tests detected significant impairments (versus SHAM), but only a few (e.g., staircase) frequently distinguished among ICH groups and none consistently differentiated among all ICH groups. However, by using a battery of tests we could behaviorally distinguish groups. Thus, preclinical testing would benefit from using a battery of behavioral tests as anything less may miss treatment effects. Such testing must be based on factors including the type of lesion, the postoperative delay and the time required to complete testing.