Intra-arterial (IA) delivery of mesenchymal stem cells (MSCs) for acute ischemic stroke is attractive for clinical translation. However, studies using rat model of stroke have demonstrated that IA MSCs delivery can decrease middle cerebral artery (MCA) flow, which may limit its clinical translation. The goal of this study is to identify a dose of IA MSCs (maximum tolerated dose; MTD) that does not compromise MCA flow and evaluate its efficacy and optimal timing in a rat model of reversible middle cerebral artery occlusion (rMCAo). We sought to determine if there is a difference in efficacy of acute (1 h) versus sub-acute (24 h) IA MSCs treatment after rMCAo. Adult female Sprague-Dawley rats underwent rMCAo (90 min) and an hour later a single dose of MSCs (at de-escalating doses 1 × 10(6), 5 × 10(5), 2 × 10(5), 1 × 10(5) and 5 × 10(4)) was given using IA route. MSCs were suspended in phosphate buffered saline (PBS) and PBS alone was used for control experiments. We measured the percent change in mean laser Doppler flow signal over the ipsilateral MCA in de-escalating doses groups to determine MTD. The results demonstrated that the lowering of IA MSC dose to 1 × 10(5) and below did not compromise MCA flow and hence an IA MSC dose of 1 × 10(5) considered as MTD. Subsequently, 1 h and 24 h after rMCAo, rats were treated with IA MSCs or PBS. The 24 h delivery of IA MSCs significantly improved neurodeficit score and reduced the mean infarct volume at one month as compared to control, but not the 1 h delivery. Overall, this study suggests that the IA delivery of MSCs can be performed safely and efficaciously at the MTD of 1 × 10(5) delivered at 24 hours in rodent model of stroke.

INTRODUCTION: There are multiple pre-clinical studies showing significant neurological functional benefit of Mesenchymal Stem Cells (MSCs) in cerebral ischemia. MSCs are adult bone marrow derived cells that have a high potential for clinical translation. The optimal route of MSC delivery has not been established. Intra-carotid (IC) delivery via catheters has several attributes attractive for clinical application & may be superior to intravenous (IV) delivery as it circumvents systemic trapping of cells and allows more cells to reach the target lesion. In our previous study using the rat rMCAo model, we found the maximum tolerated dose (MTD) of IC MSCs to be 1 x 10^5 HYPOTHESIS: We tested the hypothesis that the MTD of IC mesenchymal stem cells (MSCs) is more efficacious as compared to IV MSCs and IC control. We further hypothesized that the MTD of IC MSCs given at 24 hours is more efficacious as compared to IC MSCs at 60 minutes. METHODS: 34 female Sprague-Dawley rats underwent 90 minutes reversible middle cerebral artery occlusion (rMCAo). At 60 minutes or 24 hours post rMCAo, rats were assigned to receive: allogeneic IC MSCs at a dose of 1x 10^5 or IC phosphate buffered saline (PBS) 0.5ml or allogeneic intravenous (IV) MSCs 1 x 10^6 & rMCAo with no treatment (sham). Primary outcome measures were blinded neurodeficit score and infarct volume at 4 weeks. RESULTS: At 24 hours post rMCAo, there was no significant difference in the neurodeficit score (NDS) amongst the groups. At four weeks post treatment, the IC MSC group showed a significantly lower NDS (7%_3.3) as compared to IV MSCs (10.2%_1.5, p=0.042); In subgroup analysis, IC MSCs given at 60 minutes showed no significant difference compared to other groups, but IC MSC given at 24 hours showed a significantly lower neurodeficit score (5.8 %_2.6) as compared to IC PBS (10.8%_1.2, p=0.003), IV MSCs (10.2 %_1.5, p=0.005) and Sham (10%_ 3.4, p=0.018) ( Fig1 .). The mean infarct volume of the group treated with IC MSCs was significantly lower as compared to IC PBS (18cc%_ 8 vs. 50cc%_17, p=0.043). On infarct topography frequency map comparison, we found a significantly decreased volume of infarction in the cortical regions in IC MSC group as compared to IC PBS group. CONCLUSIONS: Treatment with maximum tolerated IC MSC dose of 1 x 10^5 post rMCAO results in superior functional outcome when compared with IV MSC 1 x 10^6, IC PBS and sham rats. Additionally, IC MSC at 24 hours is more efficacious than IC MSC at 60 minutes. There is also a significant decrease in infarct volume in IC MSC treated rats when compared with IC PBS. The IC MSCs may ameliorate injury in the cortex surrounding the core.

Plants manufacture their own compounds to protect themselves against dangers and illness. Those defenders turn out to protect humans against diseases. Chinese herbs have been used to treat humans' illness for thousands of years. This chapter reports that one specific nine-herb combination, believed to be effective in mitigating ischemic stroke injury in patients in China, protected the brain from focal and global ischemia in animal studies, demonstrated by behavioral and histopathological tests. Daily administration of the nine-herb cocktail soup by gavage, initiated at 4 hours after ischemia, reduced the infarct volume by 53 and 62%, compared to nontreated groups, at days 3 and 28, encouraged neurological function recovery, prevented brain cavitation and enhanced the self-repair in brain infarcts; the therapy reduced neuronal death in hippocampal CA1 and striatum in the acute stage, prevented the delayed encephalopathy and improved coordination at the chronic stage after global ischemia. This chapter documents 2 formulas and 19 single herbs which have been used applying to treat ischemia in China for centuries. All of these herbs are nontoxic. Their dosages and disease-fighting compounds are depicted.

Background: Bedside herbal therapy has been using in stroke patients in China for generations. The present study used a specific combination of 9 herbs including Huang Qin (Secutellaria baicalensis) in animal experiment to investigate if and how it works. The therapeutic strategies are encouraging blood circulation and self-healing, inhibiting inflammation and scavenging free oxygen radicals. Methods: Cerebral infarction was produced in overnight-fasted Male Sprague-Dawley rats (280-315g) by 2-h intraluminal occlusion in middle cerebral artery (MCA) with a suture. Head temperature was maintained at 36-36.5oC and body temperature at 37-37.5°C. All rats received behavioral test. Rats with full neurological deficit scores ranging10 -12 at 2h after MCAo participated in the study. Rats survived for 3 or 28 days before histopathological analysis. Treated rats had the herb cocktail-style (HC) soup, which was initiated at 2 hrs after reperfusion and daily administrated by gavage. Nontreated rats received drink water (DW). Results: 1. Infarct sizes were 194 ± 70 (DW, n=10) vs. 91 ± 69 mm3 (HC, n=9) on day 3; 101 ± 51 (DW, n=9) vs. 38 ± 22 mm3 (HC, n=11) on day 28. Infarct volumes decreased by 53 ? 62 % in HC groups (mean ± S.D.; ANOVA, p?0.01). Protection was in penumbra in cortex. On day 28, 8 of 9 DW rats showed cavitation; 3 of 11 HC rats had cavities. Prominent and huge cavities were in the infarcts in DW rats while small ones in HC rats. 2. Inflammation in infarcts was attenuated in HC group. Numbers of neutrophils were 548 ± 252 (HC) vs.1343 ± 431 (DW) (p=0.008); macrophages were 4485 ± 1487 (HC) vs. 7374±1824 (DW) (p<0.05) in bregma level ∼ -0.3 mm on day 3. 3. Normal endothelia (NE) in infarcted hemisphere decreased in DW group on day 3. However, amounts of NE in parietal and cingulate cortex were normal in HC rats. The occupied field-areas by NE profiles were 2.1 ± 1 % (HC) vs. 1 ± 1 % (DW) (p=.01) in parietal II region and 1.6 ± .03 % (HC) vs. 1.1 ± .05 % in cingulate cortex (DW) (p=.01). On day 28, NE were 0.8 ± 0.4 % (HC) vs. 0.3 ± 0.2 % (DW) in the infarct in striata (p=0.001). NE increased in striata in HC group, but not DW rats. Normal blood vessels were existed in the infarct core in HC rats while absent in DW rats. 4. Neurogenesis: on day 28, the numbers of newborn neurons detected by anti-βIII tubulin in peri-infarct zone and inside infarct were 143 ± 98 (HC) vs. 56 ± 48 (DW) (p=0.02) [sum of the neurons in 3 standard levels (bregma levels ∼ 2.7, -0.3 and -3.8 mm)]. 5. Neuronal deficit recovered earlier and faster in HC rats; significant recovery began at day 1 and no paralysis on day 28 while DW rats recovered later and with some deficits on day 28 (p<0.05). Conclusion: The 3-28 days of 9-herbal therapy protected brain from focal ischemia and encouraged post-ischemic neuronal function recovery and brain self-repair.

We characterized acute intracerebral hemorrhage (ICH) in the rat by sequential magnetic resonance imaging (MRI) and correlated MRI findings with neurobehavior and histopathology. In addition, we investigated whether albumin treatment would reduce ICH-induced brain injury. ICH was produced in rats by a double-injection method in which 45 microl of fresh arterial blood was injected into the right striatum. Susceptibility-weighted (SWI) and T2-weighted (T2WI) MRI was carried out on a 4.7T magnet at 0-1 h, 6 h, 24 h, 72 h, and 7 days after ICH. Animals were treated with either 25% human albumin, 1.25 g/kg, or saline vehicle i.v. at 90 min after ICH. Neurological status was evaluated before ICH and after treatment (at 4 h, 24 h, 48 h, 72 h, and 7 days). Brains were then perfusion-fixed, re-imaged on an 11.7T magnet, and studied by histopathology and immunochemistry. MRI revealed a consistent hematoma involving the striatum and overlying corpus callosum, with significant volume changes over time. Lesion volumes computed from T2WI images and by histopathology agreed closely with one another and were highly correlated (p=0.002). SWI lesion volumes were also highly correlated to histological volumes (p<0.001) but overestimated histological hematoma volume by approximately 5-fold. Albumin treatment significantly improved neurological scores compared to saline at 72 h (3.8+/-0.6 vs. 1.5+/-0.7) and 7 days (3.8+/-0.4 vs. 1.3+/-0.5, respectively, p<0.05), but did not affect histological or MRI lesion volumes. Taken together, sequential MRI plus histopathology provides a comprehensive characterization of experimental ICH. Albumin treatment improves neurological deficit after ICH but does not affect MRI or histological hematoma size.

We have previously shown that 10- and 12.5-min (but not 7-min) of global ischemia induces secondary brain damage, including incomplete infarcts in striatum and hippocampal CA.1 sector associated with endothelial degeneration and microvascular thrombosis over 4–10 weeks. 1–3 12.5-min ischemia also induces cerebral amyloid b-peptide (Ab) deposition at 8–10 weeks, concentrated in the basal cortex. Peri-vascular neuronal shrinkage degeneration coincides with evident Ab deposition.2, 3 The present study investigated endothelial degeneration in cortex in an effort to understand the relationship to brain Ab deposition.

Immunochemical staining techniques are commonly used to assess neuronal, astrocytic and microglial alterations in experimental neuroscience research, and in particular, are applied to tissues from animals subjected to ischemic stroke. Immunoreactivity of brain sections can be measured from digitized immunohistology slides so that quantitative assessment can be carried out by computer-assisted analysis. Conventional methods of analyzing immunohistology are based on image classification techniques applied to a specific anatomic location at high magnification. Such micro-scale localized image analysis limits one for further correlative studies with other imaging modalities on whole brain sections, which are of particular interest in experimental stroke research. This report presents a semi-automated image analysis method that performs convolution-based image classification on micro-scale images, extracts numerical data representing positive immunoreactivity from the processed micro-scale images and creates a corresponding quantitative macro-scale image. The present method utilizes several image-processing techniques to cope with variances in intensity distribution, as well as artifacts caused by light scattering or heterogeneity of antigen expression, which are commonly encountered in immunohistology. Micro-scale images are composed by a tiling function in a mosaic manner. Image classification is accomplished by the K-means clustering method at the relatively low-magnification micro-scale level in order to increase computation efficiency. The quantitative macro-scale image is suitable for correlative analysis with other imaging modalities. This method was applied to different immunostaining antibodies, such as endothelial barrier antigen (EBA), lectin, and glial fibrillary acidic protein (GFAP), on histology slides from animals subjected to middle cerebral artery occlusion by the intraluminal suture method. Reliability tests show that the results obtained from immunostained images at high magnification and relatively low magnification are virtually the same.

In addition to producing acute neuronal necrosis within selectively vulnerable brain regions, our recent studies have shown that global cerebral ischemia may also be followed by protracted degenerative changes occurring over the course of 10 weeks. Chronic brain pathology may be associated with the abnormal deposition of β-amyloid precursor protein (βAPP). In the present study, we used a monoclonal antibody to the N-terminal portion of βAPP to characterize the brains of rats surviving 1–10 weeks following 10 min of global brain ischemia produced by bilateral carotid artery occlusions plus systemic hypotension. After ischemia, increased βAPP immunolabeling emerged in several brain regions. In the hippocampus, granular deposits appeared in the damaged CA1 area by 2 weeks, and by 4–10 weeks the remnants of necrotic CA1 neurons were also immunolabeled. In striatum and thalamus, regions with necrotic cell death also revealed granular βAPP deposits. The neocortex was devoid of overt ischemic neuronal damage but revealed prominent βAPP immunoreactivity. Large ovoid deposits of low-density βAPP immunostaining occurred in cortical neurons at 1–2 weeks. At 4–10 weeks, large round or oval deposits immunoreactive for βAPP appeared in several cortical regions. The highest density of deposits was seen in the temporal and piriform cortices. Our results indicate that abnormal βAPP deposition may result from ischemic as well as chronic neurodegenerative processes.

Recent experimental investigations have emphasized the importance of assessing both acute and chronic histopathological changes occurring after cerebral ischemia. The purpose of this study was to evaluate the temporal profile of neuronal, astrocytic and microglial alterations within vulnerable regions (striatum and CA1 sector of hippocampus) following transient global ischemia. Anesthetized Wistar rats underwent 10 min of normothermic (37 degrees C) ischemia induced by bilateral carotid ligations plus hypotension (45-50 mm Hg) and were allowed to survive for periods ranging from 1 to 10 weeks (n=4-6/group) prior to quantitative histopathological analysis. Adjacent sections were examined by hematoxylin-and-eosin histopathology, immunostaining for glial fibrillary acidic protein, and B4-isolectin immunochemistry for microglia. In the striatum, normal-neuron counts were first decreased significantly at 2 weeks after the ischemic insult. Neuronal loss was associated with the proliferation of reactive microglia, which peaked at 1 week. By contrast, reactive astrocytosis displayed a more protracted pattern, with peak activation at 2 weeks. In the CA1 hippocampus, a decreased number of normal neurons was seen at 1 week post ischemia, together with a significant increase in immunoreactive microglia at that time; the latter normalized after 2 weeks. Reactive astrocytes in the CA1 hippocampus were significantly increased at 1-2 weeks after ischemia. In a subgroup of severely injured animals, foci of frank striatal infarction were associated with early and severe microglial and astrocytic proliferation at week 4 or later. Finally, cerebrovascular changes included endothelial disruption within affected areas. These observations document a subacute and chronic sequence of cellular responses following brief periods of global ischemia, involving both neurons, glia and vascular endothelium.

Ludmila Belayev, Weizhao Zhao, Pradip M. Pattany, R. Greg Weaver, Pil W. Huh, Baowan Lin, Efficacy of Albumin Therapy in Focal Cerebral Ischemia Diffusion-Weighted Magnetic Resonance Imaging Confirms Marked Neuroprotective Print ISSN: 0039-2499. Online ISSN: 1524-4628 Copyright © 1998 American Heart Association, Inc. All rights reserved. is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Stroke doi: 10.1161/01.STR.29.12.2587 1998;29:2587-2599 Stroke. http://stroke.ahajournals.org/content/29/12/2587 World Wide Web at: The online version of this article, along with updated information and services, is located on the

We undertook a detailed characterization of the cellular responses to acute global cerebral ischemia complicated by hyperglycemia. Anesthetized, physiologically monitored male Wistar rats received 12.5 min of global forebrain ischemia by bilateral common carotid artery occlusions plus hemorrhagic hypotension to 45 mmHg. Cranial temperature was maintained at normothermic levels. Hyperglycemic animals received dextrose (2.5 ml of a 25% solution, intraperitoneally) prior to ischemia; this doubled the mean plasma glucose concentration to 296 mg/100 ml. At 3 days (n = 10) or 24 h (n = 4) after ischemia, brains were perfusion-fixed and paraffin-embedded for light microscopic histopathology and for the histochemical visualization of activated microglia and the immunocytochemical visualization of glial fibrillary acid protein. Normal-neuron counts in the vulnerable hippocampal CA1 sector of hyperglycemic-ischemic (HI) rats were reduced to one-third the number observed in normoglycemic-ischemic (NI) animals. Ischemic cell counts in the striatum were increased fivefold or more in HI compared to NI rats, and normal small-neuron counts were reduced by two-thirds. The neocortex and striatum of NI rats showed only mild damage, while the majority of HI rats had extensive lesions, and several showed large cortical, striatal or thalamic infarcts. In addition, widespread cortical ischemic neuronal changes were evident in HI animals. No endothelial alterations were present in NI rats. By contrast, HI rats showed prominent peri- and intravascular polymorphonuclear and monocytic accumulation evident at 24 h; frequent white cell thrombi in pial arterioles on day 3; and thickening of vascular endothelium, with foci of parenchymal rarefaction or microinfarction adjacent to occluded vessels. Prominent microglial activation, often along the course of penetrating blood vessels, was common in the striatum and neocortex of HI animals but was much less extensive in the NI group. Activated microglia in HI rats were typically hypertrophic and amoeboid. These results suggest that the detrimental influence of hyperglycemia in ischemia is initially mediated by an action on vascular endothelium, which in turn leads to widespread foci of infarction and neuronal loss.