Search

Introduction: Cell-based therapies for stroke remain under development and there is a lack of pre-clinical large animal data defining optimal dosing and delivery methods. Whereas safety and efficacy of intra-arterial (IA) mesenchymal stem cells (MSC) have been demonstrated in rodent models, large animal model are lacking, hampering advancement of the field and limiting mechanistic insights. Methods: An endovascular canine reversible Middle Cerebral Artery Occlusion (MCAO) model using a retractable platinum coil for 60-120 min was established. At 48-hour post-MCAO, allogeneic male canine MSCs (10-80 million) were delivered using a microcatheter in the ipsilateral upper cervical internal carotid artery of female canine subjects. Serial MRIs and neurological deficit scoring (NDS) were performed over 30 days. Animals were euthanized at15-30 d post-MCAO and brains were harvested for histology and molecular analyses. Results: There was a dose-dependent reduction of infarct volume on MRI associated with improved neurological scoring and corticospinal tract caliber on Diffusion Tensor Imaging over the range of 10, 20 and 40 million MSCs vs. PBS control. This dose-range was safe and did not result in worsening of stroke volume or behavioral outcomes. In contrast, 80 million IA MSCs led to a worsened neurological score immediately following the injection and an increase in the infarct volume at 4 days post injection. Conclusion: Together these findings establish a successful preclinical canine model of stroke in which dose-dependent efficacy of IA MSC therapy is demonstrated to reduce stroke size and augment DTI. These findings have important clinical implications in designing early-stage trials of IA delivery in humans.

Background: Despite the efficacy of thrombectomy for large vessel occlusion acute ischemic stroke (AIS) , ~50% of patients have significant residual deficits. Pre-clinical data on intra-arterially (IA) administered mesenchymal stem cells (MSCs) in stroke are promising and this approach is attractive for clinical application. While there is a concern for micro-occlusion with IA delivery due to the large size of MSCs, a dose of 1 x 10 5 MSCs given 24-48 hr in a rodent reperfusion middle cerebral artery occlusion (rMCAo) model has been shown to be safe and effective. As per STAIR recommendations, we performed a dose-escalation (DE) study of IA-MSCs in a large animal stroke model. Methods: An endovascular canine rMCAo model using retractable platinum coil for 60-120 min was established. At 48 hr post-rMCAo, allogeniec canine MSCs were delivered using a 0.0165” microcatheter in the ipsilateral upper cervical internal carotid artery in escalating doses (based on proportion of rodent to canine total cerebral blood volume). Serial MRIs and neurological deficit scoring (NDS) were performed over 30 days. Animals were euthanized at 15-30 d post-rMCAo and brains were harvested. Results: Female canines (n=13), age 12-36 months, weighing 22-26 kg received IA MSCs ranging from 5-80 x 10 6 (M). At doses of 5-40 M IA-MSCs no neurological worsening was observed. Serial NDS and stroke volume on MRI showed no increase post-IA-MSCs and actually showed progressive reduction. A higher numerical reduction was seen in the 10-40 M groups compared to 5 M. However, in the one canine receiving 80 M IA-MSCs, there was significant worsening of the MCA-area infarction and NDS due to microembolization at this higher dose. Gross examinations and histopathology of brain tissue were consistent with ischemia. The brain of a canine receiving 80 M cells showed differentially aged areas of necrosis supporting two ischemic events. Neuroblasts, doublecortin-positive cells, and neovascularization were observed in canine brains suggesting regenerative mechanisms. Conclusions: These data suggest that IA-MSCs are safe in a large animal model up to 40 M IA-MSCs and is the maximum tolerated dose in this DE study. Furthermore, our data suggests that up to 40 M IA-MSCs may be promising in exploring efficacy in AIS.

Background: Imaging biomarkers of stroke recovery are critical for translational studies. Diffusion tensor imaging (DTI) to assess white matter microstructure in the brain using fractional anisotropy (FA) to dertermine the structural integrity of the corticospinal tract (CST). Our prior data suggest a possible correlation between neurological recovery and the improvement in FA. The purpose of this study was to compare DTI analysis of the CST at 30 days after an ipsilateral intra-arterial (IA) Mesenchymal Stem Cell (MSCs) versus control, in canines with a reversible Middle Cerebral Artery occlusion (rMCAo). Methods: Mongrel Hounds (n=7), aged 12-36 months, were included in this study. rMCAo was induced via endovascular approach using a detachable helical ultra-coil over 35-80 min. MSCs (1-20 millions) or saline (240 cc) were infused intra-arterially 48hrs post stroke in the ipsilesional cervical internal carotid. Brain MRIs were performed at 48h post rMCAo pre-IA cell delivery, 15 & 30 days. FA values of the right and left CST were determined. DTT was also generated for a visual analysis of the CST fibers. Weekly neuro evaluations were performed using blinded canine stroke-score. Results: We observed a higher average change in the FA values from at 30 days post stroke in the MSCs treated group versus control (0.085 ± 0.025, n=5 versus 0.068 ± 0.058, n=2: p=0.82). A visual increase in the caliber of CST ipsilateral to stroke from IA injection to day 30 post-stroke was observed on the DTT in two of the treated canines. Figure-1 from left to right, a. FA changes, b. DTT treated group, C. DTT control. Conclusions: Serial DTI-DTT imaging after IA MSCs therapy in large animal stroke model appears to have a trend towards higher change in FA. These findings could support further development of DTI-DTT biomarkers to measure neurologic recovery in experimental models as well as early clinical trials of novel stroke therapies.

Background: Endocrine dysfunction, including thyroid function, has been known to affect cerebral vascular dysfunction. Hypothyroidism has been shown to cause mucopolysaccharide deposition which has been associated with the weakening of blood vessel walls and aneurysm formation in some case reports. However, the connection between hypothyroidism and cerebral aneurysm has not been well studied. We hypothesized that hypothyroidism maybe associated with occurrence of cerebral aneurysms. Methods: We performed a retrospective case-control study of consecutive patients receiving cerebral angiography at our tertiary-care academic medical center. Inclusion criteria included undergoing 3-vessel angiography. Exclusion criteria included any ruptured aneurysm or previous intracranial hemorrhage. Medical history of hypothyroidism was noted along with all other risk factors. The control group of patients did not have any intra-cranial abnormality on angiography. Patients with cerebral aneurysm were matched with controls for known risk factors of cerebral aneurysm, including: age, hypertension, and smoking history. Cases were further subdivided base on gender given females are known to have higher incidence of hypothyroidism. Results: From 2008 through 2013, 82 female and 59 male patients with aneurysms were identified for this study and were matched with equal numbers of controls. Among women, hypothyroidism was present in 20.7% (17/82) among patients with cerebral aneurysms vs. in only 6.1% (5/82) among matched controls: odds ratio (OR) 4.03 (95% CI 1.41-11.51, p=0.01). Among men, 8.5% (5/59) cases and 0/59 controls had hypothyroidism, showing no significant association, with an odds ratio of 12.01 (95% CI 0.65-222.3, p=0.095). Conclusion: We found a significant association of history of hypothyroidism with cerebral aneurysm in women in our patient cohort. Given the potential pathophysiologic connection between hypothyroidism and vascular wall dysfunction, this should be explored further.

Background: Cell therapy is promising in rodent pre-clinical studies. Confirmation of efficacy of this novel approach in large animals is recommended. Diffusion Tensor Imaging (DTI) assesses white matter microstructure in the CNS using Fractional anisotropy (FA) and apparent diffusion coefficient (ADC). Diffusion Tensor Tractography (DTT) DTT produces a 3D representation of the white matter tracts (WMT) in which data are displayed on a colored map that helps to identify the integrity of the tracts selected under the region of Interest (ROI). The purpose of this study was to use DTI-DTT analysis, and correlate it with the neuro-assessment of dogs over one month after ipsilateral intra-arterial (IA) Mesenchymal Stem Cell (MSCs) 48 hours after a brain-stroke was reversible middle cerebral artery occlusion (rMCAo) Methods: Mongrel Hounds (n=5), aged 12-36 months, were included in this pilot study. rMCAo was induced via endovascular approach using a detachable helical ultra-coil over 35-80 min. MSCs (1-20 millions) were infused intra-arterially 48hrs post stroke in the ipsilesional cervical internal carotid. DTI-T images were obtained at 48h post rMCAo pre-IA cell delivery, 15 & 30 days. DTT was also generated. FA-ADC values of the right & left hemisphere and CST were determined. Weekly neuro-evaluations were performed using our canine neuro-scale Results: Our data suggest a correlation between the neurological recovery and improvement in the FA-ADC values (see figure). An increase in the caliber of corticospinal tracts ipsilateral to the stroke by day 30 compared to pre-injection was observed on the DTT reconstruction for two canines that showed neurological improvement ( see figure) Conclusions: Serial DTI-DTT imaging after IA cell therapy shows improvement in white matter tracts correlating with neurologic recovery. This supports further development of DTI-DTT biomarkers to measure neurologic recovery in experimental models as well as early clinical trials.

Background: Stroke is a leading cause of major long-term disability worldwide. Despite availability of IV tPA and mechanical thrombectomy, over half of treated patients have significant disability. Cell based therapy is a promising in pre-clinical studies in improving stroke recovery. Brain infarct volume is a major predictor of stroke outcome and may decrease with this treatment. We hypothesized that patients treated with intra-arterial stem cell therapy will have a significant reduction in stroke volume compared to non-treated patients. Methods: The RECOVER-Stroke trial is a randomized, sham-controlled study to determine the safety and efficacy of processed autologous bone marrow cells (ALD-401) injection via intracarotid infusion in ischemic stroke patients. Ischemic stroke patients were double-blind randomized into 2 groups, receiving ALD-401 injection or sham procedure between 9-19 days post anterior circulation ischemic stroke. All enrolled patients at 1 of 8 sites in the trial were included in this analysis. Two blinded physicians used OSIRIX software to analyze stroke volume on DWI/ 24h CT brain and MRI at 90, 180, 365 days post intervention. Volumes were compared between the 2 groups at these timepoints. Results: A total of 11 patients at our center (out of 48 multicenter patients) were included in this study, 6 patients receiving sham procedure and 5 patients receiving ALD-401. The two groups did not show any significant difference in their baseline variables including age, Mean baseline stroke volumes were not significantly different between ALD-401 (103.9±96.2ml) and sham groups (70.2±46ml), p=0.53. The stroke volume reduction in the ALD-401 was 22.9±15.5ml vs. 4.04±12.5ml in the sham group (p=0.046). On day 180 and 365, both groups did not show any further significant stroke volume reduction from day 90. Conclusion: In this exploratory analysis from one center in the multi-center RECOVER-Stroke trial, intra-arterial stem cell injection with ALD-401 was associated with significant reduction in stroke volume at 90 days post therapy compared to sham procedure in subacute ischemic stroke patients.

Background: Cell therapy is promising in pre-clinical studies of global ischemia. The aim of this pilot study is to describe the first intra-arterial (IA) delivery of bone marrow derived ALD-401 cells in a persistent vegetative state (PVS) patient in the U.S. & findings on MRI Diffusion Tensor Imaging (DTI) and Diffusion Tensor Tractography (DTT). Material: A 51-yr. woman developed PVS due to cardiac arrest. At 2 years post PVS state, experimental cell therapy was requested by the family. The FDA approved a one-time compassionate use IA delivery of ALD-401 cells. Bone marrow harvest from the patient, followed by processing with flow cytometry was completed to separate ALD-401 cells. Intra-arterial infusion of cells was done bilaterally into each supra-clinoid carotid. Post-infusion neurological assessments- National Institute of Health Stroke Scale (NIHSS), JFKr coma recovery scale, brainstem reflexes, modified Rankin Scores (mRS) and MRI DTI imaging were collected at 1-, 3-, 6-, 9-mo & 1 year Result: The patient had no adverse events peri-operatively. Over 1-year follow-up the patient had 1 hospitalization for UTI and postmenopausal bleeding. No other adverse events occurred. Minor clinical improvement was shown on assessment scales above. However, DTI-DTT analysis of Fractional Anisotropy (FA) showed improvement in the FA and ADC of white matter tracts starting 7 months post injection for the Cortical Spinal Tract (CST) and starting 4 months post injection for the Corpus Callosum (CC) months (see figure). Diffusion Tensor Tractography also showed qualitative improvement in organization and caliber of CC & CST over follow-up post intervention (see figure) Conclusion: Assessing minor neurological improvements in this severe PVS patient remains challenging; DTI-DTT MRI imaging detects changes in white mater tracts that could serve as biomarkers for recovery. Further follow-up clinical assessments and imaging will continue in this patient to define further recovery.