Vasculature Remodeling in a Rat Model of Cerebral Ischemia. The Fate of the BrdU-Labeled Cells Prior to Stroke

Despite the clinical significance of post-stroke angiogenesis, a detailed phenotypic analysis of pre-stroke vascular remodeling and post-stroke angiogenesis had not yet been done in a model of focal ischemia. In this study, using BrdU-labeling of proliferating cells and immunofluorescence of pre- and post-stroke rats, we found that, (i) BrdU administered before stroke was incorporated preferentially into the nuclei of endothelial cells lining the lumen of existing blood vessels and newly born neurons in the dentate gyrus but not in the subventricular zone or proliferating microglia, (ii) BrdU injection prior to stroke led to the patchy distribution of the newly incorporated endothelial cells into existing blood vessels of the adult rat brain, (iii) BrdU injection prior to stroke specifically labeled neuronal precursors cells in a region of soft tissue beyond the inhibitory scar, which seems to be permissive to regenerative events, (iv) BrdU injection after stroke led to labeling of endothelial cells crossing or detaching from the disintegrating blood vessels and their incorporation into new blood vessels in the stroke region, scar tissue and the region beyond, (v) BrdU injection after stroke led to specific incorporation of BrdU-positive nuclei into the “pinwheel” architecture of the ventricular epithelium, (vi) blood vessels in remote areas relative to the infarct core and in the contralateral non-lesioned cortex, showed co-labeled BrdU/RECA+ endothelial cells shortly after the BrdU injection, which strongly suggests a bone marrow origin of the endothelial cells. In the damaged cortex, a BrdU/prolyl 4-hydroxylase beta double labeling in the close proximity to collagen IV-labeled basement membrane, suggests that, in addition to bone marrow derived endothelial cells, the disintegrating vascular wall itself could also be a source of proliferating endothelial cells, (vii) By day 28 after stroke, new blood vessels were observed in the perilesional area and the scar tissue region, which is generally considered to be resistant to regenerative events. Finally, (viii) vigorous angiogenesis was also detected in a region of soft tissue, also called “islet of regeneration,” located next to the inhibitory scar.Conclusion: BrdU administered prior to, and after stroke, allows to investigate brain vasculature remodeling in the adult brain

Protective Effect of Anthocyanin on Neurovascular Unit in Cerebral Ischemia/Reperfusion Injury in Rats

Treating cerebral ischemia continues to be a clinical challenge. Studies have shown that the neurovascular unit (NVU), as the central structural basis, plays a key role in cerebral ischemia. Here, we report that anthocyanin, a safe and natural antioxidant, could inhibit apoptosis and inflammation to protect NVU in rats impaired by middle cerebral artery occlusion/reperfusion (MCAO/R). Administration of anthocyanin significantly reduced infarct volume and neurological scores in MCAO/R rats. Anthocyanin could also markedly ameliorate cerebral edema and reduce the concentration of Evans blue (EB) by inhibiting MMP-9. Moreover, anthocyanin alleviated apoptotic injury resulting from MCAO/R through the regulation of Bcl-2 family proteins. The levels of inflammation-related molecules including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6), which were over-expressed with MCAO/R, were decreased by anthocyanin. In addition, Nuclear factor-kappa B (NF-κB) and the NLRP3 inflammasome pathway might be involved in the anti-inflammatory effect of anthocyanin. In conclusion, anthocyanin could protect the NVU through multiple pathways, and play a protective role in cerebral ischemia/reperfusion injury

Oophorectomy Reduces Estradiol Levels and Long-Term Spontaneous Neurovascular Recovery in a Female Rat Model of Focal Ischemic Stroke

Although epidemiological evidence suggests significant sex and gender-based differences in stroke risk and recovery, females have been widely under-represented in preclinical stroke research. The neurovascular sequelae of brain ischemia in females, in particular, are largely uncertain. We set out to address this gap by a multimodal in vivo study of neurovascular recovery from endothelin-1 model of cortical focal-stroke in sham vs. ovariectomized female rats. Three weeks post ischemic insult, sham operated females recapitulated the phenotype previously reported in male rats in this model, of normalized resting perfusion but sustained peri-lesional cerebrovascular hyperreactivity. In contrast, ovariectomized (Ovx) females showed reduced peri-lesional resting blood flow, and elevated cerebrovascular responsivity to hypercapnia in the peri-lesional and contra-lateral cortices. Electrophysiological recordings showed an attenuation of theta to low-gamma phase-amplitude coupling in the peri-lesional tissue of Ovx animals, despite relative preservation of neuronal power. Further, this chronic stage neuronal network dysfunction was inversely correlated with serum estradiol concentration. Our pioneering data demonstrate dramatic differences in spontaneous recovery in the neurovascular unit between Ovx and Sham females in the chronic stage of stroke, underscoring the importance of considering hormonal-dependent aspects of the ischemic sequelae in the development of novel therapeutic approaches and patient recruitment in clinical trials

Multiparametric measurement of cerebral physiology using calibrated fMRI

The ultimate goal of calibrated fMRI is the quantitative imaging of oxygen metabolism (CMRO2), and this has been the focus of numerous methods and approaches. However, one underappreciated aspect of this quest is that in the drive to measure CMRO2, many other physiological parameters of interest are often acquired along the way. This can significantly increase the value of the dataset, providing greater information that is clinically relevant, or detail that can disambiguate the cause of signal variations. This can also be somewhat of a double-edged sword: calibrated fMRI experiments combine multiple parameters into a physiological model that requires multiple steps, thereby providing more opportunity for error propagation and increasing the noise and error of the final derived values. As with all measurements, there is a trade-off between imaging time, spatial resolution, coverage, and accuracy. In this review, we provide a brief overview of the benefits and pitfalls of extracting multiparametric measurements of cerebral physiology through calibrated fMRI experiments

Microglia at center stage: a comprehensive review about the versatile and unique residential macrophages of the central nervous system

Microglia cells are the unique residential macrophages of the central nervous system (CNS). They have a special origin, as they derive from the embryonic yolk sac and enter the developing CNS at a very early stage. They play an important role during CNS development and adult homeostasis. They have a major contribution to adult neurogenesis and neuroinflammation. Thus, they participate in the pathogenesis of neurodegenerative diseases and contribute to aging. They play an important role in sustaining and breaking the blood-brain barrier. As innate immune cells, they contribute substantially to the immune response against infectious agents affecting the CNS. They play also a major role in the growth of tumours of the CNS. Microglia are consequently the key cell population linking the nervous and the immune system. This review covers all different aspects of microglia biology and pathology in a comprehensive way

Microglia at center stage: a comprehensive review about the versatile and unique residential macrophages of the central nervous system

Microglia cells are the unique residential macrophages of the central nervous system (CNS). They have a special origin, as they derive from the embryonic yolk sac and enter the developing CNS at a very early stage. They play an important role during CNS development and adult homeostasis. They have a major contribution to adult neurogenesis and neuroinflammation. Thus, they participate in the pathogenesis of neurodegenerative diseases and contribute to aging. They play an important role in sustaining and breaking the blood-brain barrier. As innate immune cells, they contribute substantially to the immune response against infectious agents affecting the CNS. They play also a major role in the growth of tumours of the CNS. Microglia are consequently the key cell population linking the nervous and the immune system. This review covers all different aspects of microglia biology and pathology in a comprehensive way

Cellular interactions during neural repair

Injury to the central nervous system induces a limited neural repair response that is causally linked to recovery of function. The goal of this dissertation is to better understand neural repair responses, including how different cell types respond to injury and how cellular interactions shape repair. I investigate repair processes in a mouse model of cortical stroke by applying behavioral, imaging, and genetic techniques. In Chapter 2, I characterize the spatiotemporal dynamics of vascular plasticity following stroke, and its association with restoration of blood flow and behavioral function. I find that stroke instigates a short time window during which vascular plasticity unfolds. This window of vascular plasticity is underpinned by transient activation of pro-angiogenic gene expression programs. The formation of new vessels is associated with the restoration of blood flow to peri-infarct regions, which is in turn associated with behavioral improvement. In Chapter 3, I examine reactive astrocyte responses after stroke and their interaction with vascular plasticity. I find that stroke triggers gene expression and functional changes in reactive astrocytes that enable them to support vascular remodeling and repair. Astrocytes orchestrate multiple aspects of vascular repair, including reorganization of extracellular matrix and pericyte attachment to vessels. Ablating reactive astrocytes results in reduced angiogenesis, prolonged blood flow deficits, increased vascular permeability, and worse functional recovery. In Chapter 4 I investigate the subventricular zone (SVZ) cytogenic response to stroke. I find that cells arising from the SVZ after stroke are predominantly undifferentiated precursors and astrocytes. Arrest of cytogenesis by ablation of neural stem cells or aging reduces behavioral recovery. SVZ cytogenesis provides trophic support via vascular endothelial growth factor (VEGF) that drives effective synaptic and vascular plasticity to improve recovery. Replacement of VEGF in peri-infarct cortex of mice lacking cytogenesis is sufficient to increase dendritic spine and vascular density and enhance recovery. Together, these studies refine our understanding of how different cell types response to injury and how cellular interactions coordinate neural repair.Neuroscienc

Modulation of interhemispheric inhibition and its effect on stroke recovery in the rat

Rationale: Residual disability following stroke is a significant challenge for patients and the healthcare system, both in New Zealand and worldwide. Poor recovery from motor cortex (M1) stroke has been associated with an increase in interhemispheric inhibition (IHI) initiated by the unlesioned cortex (Murase, Duque, Mazzocchio, & Cohen, 2004). Previously we showed using intracellular recordings from single neurons that intermittent theta-burst stimulation (iTBS; bursts of three pulses at 50 Hz at 5 Hz) delivered to the contralateral M1 acutely reduced IHI in normal rats (Barry, Boddington, Igelström, et al., 2014). Additionally, this stimulation improved motor recovery sub acutely following stroke (Boddington, Gray, Schulz, & Reynolds, 2020). In the present study we aimed to record IHI changes in the freely moving, stroke affected rat and measure any association with behavioural impairment. We also aimed to determine if iTBS could modify IHI and improve behaviour. Method: A method of measuring IHI in rats using EEG recording techniques was first developed. Second, to study effects of stroke and iTBS, lesions were induced by injecting endothelin-1 into M1 and dorsal sub-cortical regions and electrodes implanted to measure IHI in freely moving animals. Sham stimulation or iTBS was delivered at low intensity to the contralesional cortex for 15 days, followed by three weeks of no stimulation. Behaviour was recorded throughout, using grid walking and pasta handling tasks. Results: Baseline IHI was 19.1% ± 4.4 and was increased to 34.3% ± 5.5 one week following lesion induction (mean difference: 15.2 ± 6.0, p=0.033). In addition, increased IHI was weakly associated with worsened motor performance (R2: 0.11, p<0.005). Comparing iTBS and sham stimulated animals, in the final week of the study, iTBS animals showed a 54% ± 8.7 functional improvement in foot faults from their post stroke grid walking impairment, compared to only 15% ± 11 in the sham group; mixed effects model: p<0.05. The pasta handling task also revealed significantly greater improvement following iTBS (improvement of 56% ± 17) compared to sham (14% ± 7.4): p<0.05. In addition, delivery of iTBS reduced IHI to a greater degree than the spontaneous reduction observed in the sham group (linear mixed model: p<0.05), both in the weeks during and following stimulation. Conclusion: Our results show an association between IHI and behavioural recovery in the early stages of stroke recovery. They also show that IHI is reduced, and behavioural recovery improved by low intensity, contralesional iTBS