ChABC infusions into medial prefrontal cortex, but not posterior parietal cortex, improve the performance of rats tested on a novel, challenging delay in the touchscreen TUNL task.

Perineuronal nets (PNNs) are specialized extracellular matrix structures that surround subsets of neurons throughout the central nervous system (CNS). They are made up of chondroitin sulfate proteoglycans (CSPGs), hyaluronan, tenascin-R, and many other link proteins that together make up their rigid and lattice-like structure. Modulation of PNNs can alter synaptic plasticity and thereby affect learning, memory, and cognition. In the present study, we degraded PNNs in the medial prefrontal (mPFC) and posterior parietal (PPC) cortices of Long-Evans rats using the enzyme chondroitinase ABC (ChABC), which cleaves apart CSPGs. We then measured the consequences of PNN degradation on spatial working memory (WM) with a trial-unique, non-matching-to location (TUNL) automated touchscreen task. All rats were trained with a standard 6 sec delay and 20 sec inter-trial interval (ITI) and then tested under four different conditions: a 6 sec delay, a variable 2 or 6 sec delay, a 2 sec delay with a 1 sec ITI (interference condition), and a 20 sec delay. Rats that received mPFC ChABC treatment initially performed TUNL with higher accuracy, more selection trials completed, and fewer correction trials completed compared to controls in the 20 sec delay condition but did not perform differently from controls in any other condition. Rats that received PPC ChABC treatment did not perform significantly differently from controls in any condition. Posthumous immunohistochemistry confirmed an increase in CSPG degradation products (C4S stain) in the mPFC and PPC following ChABC infusions while WFA staining intensity and parvalbumin positive neuron number were decreased following mPFC, but not PPC, ChABC infusions. These findings suggest that PNNs in the mPFC play a subtle role in spatial WM, but PNNs in the PPC do not. Furthermore, it appears that PNNs in the mPFC are involved in adapting to a challenging novel delay, but that they do not play an essential role in spatial WM function.

An Overview of Animal Models Related to Schizophrenia.

Schizophrenia is a heterogeneous psychiatric disorder that is poorly treated with current therapies. In this brief review, we provide an update regarding the use of animal models to study schizophrenia in an attempt to understand its aetiology and develop novel therapeutic strategies. Tremendous progress has been made developing and validating rodent models that replicate the aetiologies, brain pathologies, and behavioural abnormalities associated with schizophrenia in humans. Here, models are grouped into 3 categories-developmental, drug induced, and genetic-to reflect the heterogeneous risk factors associated with schizophrenia. Each of these models is associated with varied but overlapping pathophysiology, endophenotypes, behavioural abnormalities, and cognitive impairments. Studying schizophrenia using multiple models will permit an understanding of the core features of the disease, thereby facilitating preclinical research aimed at the development and validation of better pharmacotherapies to alter the progression of schizophrenia or alleviate its debilitating symptoms.

Enhancing Spinal Plasticity Amplifies the Benefits of Rehabilitative Training and Improves Recovery from Stroke.

The limited recovery that occurs following stroke happens almost entirely in the first weeks postinjury. Moreover, the efficacy of rehabilitative training is limited beyond this narrow time frame. Sprouting of spared corticospinal tract axons in the contralesional spinal cord makes a significant contribution to sensorimotor recovery, but this structural plasticity is also limited to the first few weeks after stroke. Here, we tested the hypothesis that inducing plasticity in the spinal cord during chronic stroke could improve recovery from persistent sensorimotor impairment. We potentiated spinal plasticity during chronic stroke, weeks after the initial ischemic injury, in male Sprague-Dawley rats via intraspinal injections of chondroitinase ABC. Our data show that chondroitinase injections into the contralesional gray matter of the cervical spinal cord administered 28 d after stroke induced significant sprouting of corticospinal axons originating in the peri-infarct cortex. Chondroitinase ABC injection during chronic stroke without additional training resulted in moderate improvements of sensorimotor deficits. Importantly, this therapy dramatically potentiated the efficacy of rehabilitative training delivered during chronic stroke in a skilled forelimb reaching task. These novel data suggest that spinal therapy during chronic stroke can amplify the benefits of delayed rehabilitative training with the potential to reduce permanent disability in stroke survivors.SIGNIFICANCE STATEMENT The brain and spinal cord undergo adaptive rewiring ("plasticity") following stroke. This plasticity allows for partial functional recovery from stroke induced sensorimotor impairments. However, the plasticity that underlies recovery occurs predominantly in the first weeks following stroke, and most stroke survivors are left with permanent disability even after rehabilitation. Using animal models, our data show that removal of plasticity-inhibiting signals in the spinal cord (via intraspinal injections of the enzyme chondroitinase ABC) augments rewiring of circuits connecting the brain to the spinal cord, even weeks after stroke. Moreover, this plasticity can be harnessed by rehabilitative training to significantly promote sensorimotor recovery. Thus, intraspinal therapy may augment rehabilitative training and improve recovery even in individuals living with chronic disability due to stroke.

Altered excitatory-inhibitory balance within somatosensory cortex is associated with enhanced plasticity and pain sensitivity in a mouse model of multiple sclerosis.

BACKGROUND: Chronic neuropathic pain is a common symptom of multiple sclerosis (MS). MOG35-55-induced experimental autoimmune encephalomyelitis (EAE) has been used as an animal model to investigate the mechanisms of pain in MS. Previous studies have implicated sensitization of spinal nociceptive networks in the pathogenesis of pain in EAE. However, the involvement of supraspinal sites of nociceptive integration, such as the primary somatosensory cortex (S1), has not been defined. We therefore examined functional, structural, and immunological alterations in S1 during the early stages of EAE, when pain behaviors first appear. We also assessed the effects of the antidepressant phenelzine (PLZ) on S1 alterations and nociceptive (mechanical) sensitivity in early EAE. PLZ has been shown to restore central nervous system (CNS) tissue concentrations of GABA and the monoamines (5-HT, NA) in EAE. We hypothesized that PLZ treatment would also normalize nociceptive sensitivity in EAE by restoring the balance of excitation and inhibition (E-I) in the CNS. METHODS: We used in vivo flavoprotein autofluorescence imaging (FAI) to assess neural ensemble responses in S1 to vibrotactile stimulation of the limbs in early EAE. We also used immunohistochemistry (IHC), and Golgi-Cox staining, to examine synaptic changes and neuroinflammation in S1. Mechanical sensitivity was assessed at the clinical onset of EAE with Von Frey hairs. RESULTS: Mice with early EAE exhibited significantly intensified and expanded FAI responses in S1 compared to controls. IHC revealed increased vesicular glutamate transporter (VGLUT1) expression and disrupted parvalbumin+ (PV+) interneuron connectivity in S1 of EAE mice. Furthermore, peri-neuronal nets (PNNs) were significantly reduced in S1. Morphological analysis of excitatory neurons in S1 revealed increased dendritic spine densities. Iba-1+ cortical microglia were significantly elevated early in the disease. Chronic PLZ treatment was found to normalize mechanical thresholds in EAE. PLZ also normalized S1 FAI responses, neuronal morphologies, and cortical microglia numbers and attenuated VGLUT1 reactivity-but did not significantly attenuate the loss of PNNs. CONCLUSIONS: These findings implicate a pro-excitatory shift in the E-I balance of the somatosensory CNS, arising early in the pathogenesis EAE and leading to large-scale functional and structural plasticity in S1. They also suggest a novel antinociceptive effect of PLZ treatment.

Cerebral collaterals and collateral therapeutics for acute ischemic stroke.

Cerebral collaterals are vascular redundancies in the cerebral circulation that can partially maintain blood flow to ischemic tissue when primary conduits are blocked. After occlusion of a cerebral artery, anastomoses connecting the distal segments of the MCA with distal branches of the ACA and PCA (known as leptomeningeal or pial collaterals) allow for partially maintained blood flow in the ischemic penumbra and delay or prevent cell death. However, collateral circulation varies dramatically between individuals, and collateral extent is significant predictor of stroke severity and recanalization rate. Collateral therapeutics attempt to harness these vascular redundancies by enhancing blood flow through pial collaterals to reduce ischemia and brain damage after cerebral arterial occlusion. While therapies to enhance collateral flow remain relatively nascent neuroprotective strategies, experimental therapies including inhaled NO, transient suprarenal aortic occlusion, and electrical stimulation of the parasympathetic sphenopalatine ganglion show promise as collateral therapeutics with the potential to improve treatment of acute ischemic stroke.

Synthesis, transport, and metabolism of serotonin formed from exogenously applied 5-HTP after spinal cord injury in rats.

Spinal cord transection leads to elimination of brain stem-derived monoamine fibers that normally synthesize most of the monoamines in the spinal cord, including serotonin (5-hydroxytryptamine, 5-HT) synthesized from tryptophan by enzymes tryptophan hydroxylase (TPH, synthesizing 5-hydroxytryptophan, 5-HTP) and aromatic l-amino acid decarboxylase (AADC, synthesizing 5-HT from 5-HTP). Here we examine whether spinal cord caudal to transection remains able to manufacture and metabolize 5-HT. Immunolabeling for AADC reveals that, while most AADC is confined to brain stem-derived monoamine fibers in spinal cords from normal rats, caudal to transection AADC is primarily found in blood vessel endothelial cells and pericytes as well as a novel group of neurons (NeuN positive and GFAP negative), all of which strongly upregulate AADC with injury. However, immunolabeling for 5-HT reveals that there is no detectable endogenous 5-HT synthesis in any structure in the spinal cord caudal to a chronic transection, including in AADC-containing vessels and neurons, consistent with a lack of TPH. In contrast, when we applied exogenous 5-HTP (in vitro or in vivo), AADC-containing vessels and neurons synthesized 5-HT, which contributed to increased motoneuron activity and muscle spasms (long-lasting reflexes, LLRs), by acting on 5-HT2 receptors (SB206553 sensitive) located on motoneurons (TTX resistant). Blocking monoamine oxidase (MAO) markedly increased the sensitivity of the motoneurons (LLR) to 5-HTP, more than it increased the sensitivity of motoneurons to 5-HT, suggesting that 5-HT synthesized from AADC is largely metabolized in AADC-containing neurons and vessels. In summary, after spinal cord injury AADC is upregulated in vessels, pericytes, and neurons but does not endogenously produce 5-HT, whereas when exogenous 5-HTP is provided AADC does produce functional amounts of 5-HT, some of which is able to escape metabolism by MAO, diffuse out of these AADC-containing cells, and ultimately act on 5-HT receptors on motoneurons.

Aberrant cortical activity, functional connectivity, and neural assembly architecture after photothrombotic stroke in mice.

Despite substantial progress in mapping the trajectory of network plasticity resulting from focal ischemic stroke, the extent and nature of changes in neuronal excitability and activity within the peri-infarct cortex of mice remains poorly defined. Most of the available data have been acquired from anesthetized animals, acute tissue slices, or infer changes in excitability from immunoassays on extracted tissue, and thus may not reflect cortical activity dynamics in the intact cortex of an awake animal. Here, in vivo two-photon calcium imaging in awake, behaving mice was used to longitudinally track cortical activity, network functional connectivity, and neural assembly architecture for 2 months following photothrombotic stroke targeting the forelimb somatosensory cortex. Sensorimotor recovery was tracked over the weeks following stroke, allowing us to relate network changes to behavior. Our data revealed spatially restricted but long-lasting alterations in somatosensory neural network function and connectivity. Specifically, we demonstrate significant and long-lasting disruptions in neural assembly architecture concurrent with a deficit in functional connectivity between individual neurons. Reductions in neuronal spiking in peri-infarct cortex were transient but predictive of impairment in skilled locomotion measured in the tapered beam task. Notably, altered neural networks were highly localized, with assembly architecture and neural connectivity relatively unaltered a short distance from the peri-infarct cortex, even in regions within 'remapped' forelimb functional representations identified using mesoscale imaging with anaesthetized preparations 8 weeks after stroke. Thus, using longitudinal two-photon microscopy in awake animals, these data show a complex spatiotemporal relationship between peri-infarct neuronal network function and behavioral recovery. Moreover, the data highlight an apparent disconnect between dramatic functional remapping identified using strong sensory stimulation in anaesthetized mice compared to more subtle and spatially restricted changes in individual neuron and local network function in awake mice during stroke recovery.

Alterations in brain network connectivity and subjective experience induced by psychedelics: a scoping review.

Intense interest surrounds current research on psychedelics, particularly regarding their potential in treating mental health disorders. Various studies suggest a link between the subjective effects produced by psychedelics and their therapeutic efficacy. Neuroimaging evidence indicates an association of changes in brain functional connectivity with the subjective effects of psychedelics. We conducted a review focusing on psychedelics and brain functional connectivity. The review focused on four psychedelic drugs: ayahuasca, psilocybin and LSD, and the entactogen MDMA. We conducted searches in databases of MEDLINE, Embase, APA PsycInfo and Scopus from inception to Jun 2023 by keywords related to functional connectivity and psychedelics. Using the PRISMA framework, we selected 24 articles from an initial pool of 492 for analysis. This scoping review and analysis investigated the effects of psychedelics on subjective experiences and brain functional connectivity in healthy individuals. The studies quantified subjective effects through psychometric scales, revealing significant experiences of altered consciousness, mood elevation, and mystical experiences induced by psychedelics. Neuroimaging results indicated alterations in the functional connectivity of psychedelics, with consistent findings across substances of decreased connectivity within the default mode network and increased sensory and thalamocortical connectivity. Correlations between these neurophysiological changes and subjective experiences were noted, suggesting a brain network basis of the psychedelics' neuropsychological impact. While the result of the review provides a potential neural mechanism of the subjective effects of psychedelics, direct clinical evidence is needed to advance their clinical outcomes. Our research serves as a foundation for further exploration of the therapeutic potential of psychedelics.

Pleiotrophin Signals Through ALK Receptor to Enhance the Growth of Neurons in the Presence of Inhibitory Chondroitin Sulfate Proteoglycans.

Chondroitin sulfate proteoglycans (CSPGs), one of the major extracellular matrix components of the glial scar that surrounds central nervous system (CNS) injuries, are known to inhibit the regeneration of neurons. This study investigated whether pleiotrophin (PTN), a growth factor upregulated during early CNS development, can overcome the inhibition mediated by CSPGs and promote the neurite outgrowth of neurons in vitro. The data showed that a CSPG matrix inhibited the outgrowth of neurites in primary cortical neuron cultures compared to a control matrix. PTN elicited a dose-dependent increase in the neurite outgrowth even in the presence of the growth inhibitory CSPG matrix, with optimal growth at 15 ng mL-1 of PTN (114.8% of neuronal outgrowth relative to laminin control). The growth-promoting effect of PTN was blocked by inhibition of the receptor anaplastic lymphoma kinase (ALK) by alectinib in a dose-dependent manner. Neurite outgrowth in the presence of this CSPG matrix was induced by activation of the protein kinase B (AKT) pathway, a key downstream mediator of ALK activation. This study identified PTN as a dose-dependent regulator of neurite outgrowth in primary cortical neurons cultured in the presence of a CSPG matrix and identified ALK activation as a key driver of PTN-induced growth.

Neutrophil dynamics and inflammaging in acute ischemic stroke: A transcriptomic review.

Stroke is among the leading causes of death and disability worldwide. Restoring blood flow through recanalization is currently the only acute treatment for cerebral ischemia. Unfortunately, many patients that achieve a complete recanalization fail to regain functional independence. Recent studies indicate that activation of peripheral immune cells, particularly neutrophils, may contribute to microcirculatory failure and futile recanalization. Stroke primarily affects the elderly population, and mortality after endovascular therapies is associated with advanced age. Previous analyses of differential gene expression across injury status and age identify ischemic stroke as a complex age-related disease. It also suggests robust interactions between stroke injury, aging, and inflammation on a cellular and molecular level. Understanding such interactions is crucial in developing effective protective treatments. The global stroke burden will continue to increase with a rapidly aging human population. Unfortunately, the mechanisms of age-dependent vulnerability are poorly defined. In this review, we will discuss how neutrophil-specific gene expression patterns may contribute to poor treatment responses in stroke patients. We will also discuss age-related transcriptional changes that may contribute to poor clinical outcomes and greater susceptibility to cerebrovascular diseases.

Distinct patterns of activity in individual cortical neurons and local networks in primary somatosensory cortex of mice evoked by square-wave mechanical limb stimulation.

Artificial forms of mechanical limb stimulation are used within multiple fields of study to determine the level of cortical excitability and to map the trajectory of neuronal recovery from cortical damage or disease. Square-wave mechanical or electrical stimuli are often used in these studies, but a characterization of sensory-evoked response properties to square-waves with distinct fundamental frequencies but overlapping harmonics has not been performed. To distinguish between somatic stimuli, the primary somatosensory cortex must be able to represent distinct stimuli with unique patterns of activity, even if they have overlapping features. Thus, mechanical square-wave stimulation was used in conjunction with regional and cellular imaging to examine regional and cellular response properties evoked by different frequencies of stimulation. Flavoprotein autofluorescence imaging was used to map the somatosensory cortex of anaesthetized C57BL/6 mice, and in vivo two-photon Ca2+ imaging was used to define patterns of neuronal activation during mechanical square-wave stimulation of the contralateral forelimb or hindlimb at various frequencies (3, 10, 100, 200, and 300 Hz). The data revealed that neurons within the limb associated somatosensory cortex responding to various frequencies of square-wave stimuli exhibit stimulus-specific patterns of activity. Subsets of neurons were found to have sensory-evoked activity that is either primarily responsive to single stimulus frequencies or broadly responsive to multiple frequencies of limb stimulation. High frequency stimuli were shown to elicit more population activity, with a greater percentage of the population responding and greater percentage of cells with high amplitude responses. Stimulus-evoked cell-cell correlations within these neuronal networks varied as a function of frequency of stimulation, such that each stimulus elicited a distinct pattern that was more consistent across multiple trials of the same stimulus compared to trials at different frequencies of stimulation. The variation in cortical response to different square-wave stimuli can thus be represented by the population pattern of supra-threshold Ca2+ transients, the magnitude and temporal properties of the evoked activity, and the structure of the stimulus-evoked correlation between neurons.

Immune Modulation as a Key Mechanism for the Protective Effects of Remote Ischemic Conditioning After Stroke.

Remote ischemic conditioning (RIC), which involves a series of short cycles of ischemia in an organ remote to the brain (typically the limbs), has been shown to protect the ischemic penumbra after stroke and reduce ischemia/reperfusion (IR) injury. Although the exact mechanism by which this protective signal is transferred from the remote site to the brain remains unclear, preclinical studies suggest that the mechanisms of RIC involve a combination of circulating humoral factors and neuronal signals. An improved understanding of these mechanisms will facilitate translation to more effective treatment strategies in clinical settings. In this review, we will discuss potential protective mechanisms in the brain and cerebral vasculature associated with RIC. We will discuss a putative role of the immune system and circulating mediators of inflammation in these protective processes, including the expression of pro-and anti-inflammatory genes in peripheral immune cells that may influence the outcome. We will also review the potential role of extracellular vesicles (EVs), biological vectors capable of delivering cell-specific cargo such as proteins and miRNAs to cells, in modulating the protective effects of RIC in the brain and vasculature.

Inflammatory Cytokine Profile and Plasticity of Brain and Spinal Microglia in Response to ATP and Glutamate.

Microglia are the primary cells in the central nervous system that identify and respond to injury or damage. Such a perturbation in the nervous system induces the release of molecules including ATP and glutamate that act as damage-associated molecular patterns (DAMPs). DAMPs are detected by microglia, which then regulate the inflammatory response in a manner sensitive to their surrounding environment. The available data indicates that ATP and glutamate can induce the release of pro inflammatory factors TNF (tumor necrosis factor), IL-1ß (interleukin 1 beta), and NO (nitric oxide) from microglia. However, non-physiological concentrations of ATP and glutamate were often used to derive these insights. Here, we have compared the response of spinal cord microglia (SM) relative to brain microglia (BM) using physiologically relevant concentrations of glutamate and ATP that mimic injured conditions in the central nervous system. The data show that ATP and glutamate are not significant modulators of the release of cytokines from either BM or SM. Consistent with previous studies, spinal microglia exhibited a general trend toward reduced release of inflammatory cytokines relative to brain-derived microglia. Moreover, we demonstrate that the responses of microglia to these DAMPs can be altered by modifying the biochemical milieu in their surrounding environment. Preconditioning brain derived microglia with media from spinal cord derived mixed glial cultures shifted their release of IL-1ß and IL-6 to a less inflammatory phenotype consistent with spinal microglia.

Association of CT-Based Hypoperfusion Index With Ischemic Core Enlargement in Patients With Medium and Large Vessel Stroke.

BACKGROUND AND OBJECTIVES: The rate of infarct core progression in patients with acute ischemic stroke is variable and affects outcome of reperfusion therapy. We evaluated the hypoperfusion index (HI) to estimate the initial rate of core progression in patients with medium vessel occlusion (MeVO) compared to large vessel occlusion (LVO) stroke and within a larger time frame since stroke onset. METHODS: Core progression was assessed in 106 patients with acute stroke and CT perfusion. Using reperfusion trial core time criteria, fast progressors had core >70 mL within 6 hours of stroke onset and slow progressors had core ≤70 mL, mismatch ≥15 mL, and mismatch to core ratio ≥1.8 within 6 to 24 hours. The relationship between HI and infarct core progression (core/time) was examined using receiver operating characteristics to determine optimal HI cutoff. The HI cutoff was then tested in the overall cohort, compared between MeVO and LVO, and evaluated in patients up to 24 hours from stroke onset to differentiate fast from slow rate of core progression. HI threshold was assessed in a second independent cohort of 110 patients with acute ischemic stroke. RESULTS: In 106 patients with acute stroke, 6.6% were fast progressors, 27.4% were slow progressors, and 66% were not classified as fast or slow progressor by reperfusion trial core time criteria. HI >0.5 was associated with fast progression and able to distinguish fast from slow progressors (area under the curve [AUC] 0.94; 95% confidence interval [CI] 0.80-0.99). In MeVO (n = 26) HI >0.5 had a core progression of 0.30 mL/min compared to 0.03 mL/min for HI ≤0.5 (p < 0.001). In LVO (n = 80), HI >0.5 had a core progression of 0.26 mL/min compared to 0.02 mL/min for HI ≤0.5 (p < 0.001). In patients not classified as fast or slow progressor by reperfusion trial criteria, those with HI >0.5 had progression rate of 0.21 mL/min compared to 0.03 mL/min for those with HI ≤0.5 (p < 0.001). Validation in a second cohort of patients with acute ischemic stroke (n = 110; MeVO = 42, LVO = 68) yielded similar results for HI >0.5 to distinguish fast and slow core progression with an AUC of 0.84 (95% CI 0.72-0.97). DISCUSSION: HI can differentiate fast from slow core progression in MeVO and LVO within the first 24 hours of acute ischemic stroke. Consideration of core progression rate at time of stroke evaluation may have implications in the selection of patients with MeVO and LVO stroke for reperfusion therapy that warrant further study.

In vivo voltage-sensitive dye imaging in adult mice reveals that somatosensory maps lost to stroke are replaced over weeks by new structural and functional circuits with prolonged modes of activation within both the peri-infarct zone and distant sites.

After brain damage such as stroke, topographically organized sensory and motor cortical representations remap onto adjacent surviving tissues. It is conceivable that cortical remapping is accomplished by changes in the temporal precision of sensory processing and regional connectivity in the cortex. To understand how the adult cortex remaps and processes sensory signals during stroke recovery, we performed in vivo imaging of sensory-evoked changes in membrane potential, as well as multiphoton imaging of dendrite structure and tract tracing. In control mice, forelimb stimulation evoked a brief depolarization in forelimb cortex that quickly propagated to, and dissipated within, adjacent motor/hindlimb areas (<100 ms). One week after forelimb cortex stroke, the cortex was virtually unresponsive to tactile forelimb stimulation. After 8 weeks recovery, forelimb-evoked depolarizations reemerged with a characteristic pattern in which responses began within surviving portions of forelimb cortex (<20 ms after stimulation) and then spread horizontally into neighboring peri-infarct motor/hindlimb areas in which depolarization persisted 300-400% longer than controls. These uncharacteristically prolonged responses were not limited to the remapped peri-infarct zone and included distant posteromedial retrosplenial cortex, millimeters from the stroke. Structurally, the remapped peri-infarct area selectively exhibited high levels of dendritic spine turnover, shared more connections with retrosplenial cortex and striatum, and lost inputs from lateral somatosensory cortical regions. Our findings demonstrate that sensory remapping during stroke recovery is accompanied by the development of prolonged sensory responses and new structural circuits in both the peri-infarct zone as well as more distant sites.

Improved collateral flow and reduced damage after remote ischemic perconditioning during distal middle cerebral artery occlusion in aged rats.

Circulation through cerebral collaterals can maintain tissue viability until reperfusion is achieved. However, collateral circulation is time limited, and failure of collaterals is accelerated in the aged. Remote ischemic perconditioning (RIPerC), which involves inducing a series of repetitive, transient peripheral cycles of ischemia and reperfusion at a site remote to the brain during cerebral ischemia, may be neuroprotective and can prevent collateral failure in young adult rats. Here, we demonstrate the efficacy of RIPerC to improve blood flow through collaterals in aged (16-18 months of age) Sprague Dawley rats during a distal middle cerebral artery occlusion. Laser speckle contrast imaging and two-photon laser scanning microscopy were used to directly measure flow through collateral connections to ischemic tissue. Consistent with studies in young adult rats, RIPerC enhanced collateral flow by preventing the stroke-induced narrowing of pial arterioles during ischemia. This improved flow was associated with reduced early ischemic damage in RIPerC treated aged rats relative to controls. Thus, RIPerC is an easily administered, non-invasive neuroprotective strategy that can improve penumbral blood flow via collaterals. Enhanced collateral flow supports further investigation as an adjuvant therapy to recanalization therapy and a protective treatment to maintain tissue viability prior to reperfusion.

Impaired Collateral Flow in Pial Arterioles of Aged Rats During Ischemic Stroke.

Cerebral collateral circulation and age are critical factors in determining outcome from acute ischemic stroke. Aging may lead to rarefaction of cerebral collaterals, and thereby accelerate ischemic injury by reducing penumbral blood flow. Dynamic changes in pial collaterals after onset of cerebral ischemia may vary with age but have not been extensively studied. Here, laser speckle contrast imaging (LSCI) and two-photon laser scanning microscopy (TPLSM) were combined to monitor cerebral pial collaterals between the anterior cerebral artery (ACA) and the middle cerebral artery (MCA) in young adult and aged male Sprague Dawley rats during distal middle cerebral artery occlusion (dMCAo). Histological analysis showed that aged rats had significantly greater volumes of ischemic damage than young rats. LSCI showed that cerebral collateral perfusion declined over time after stroke in aged and young rats, and that this decline was significantly greater in aged rats. TPLSM demonstrated that pial arterioles narrowed faster after dMCAo in aged rats compared to young adult rats. Notably, while arteriole vessel narrowing was comparable 4.5 h after ischemic onset in aged and young adult rats, red blood cell velocity was stable in young adults but declined over time in aged rats. Overall, red blood cell flux through pial arterioles was significantly reduced at all time-points after 90 min post-dMCAo in aged rats relative to young adult rats. Thus, collateral failure is more severe in aged rats with significantly impaired pial collateral dynamics (reduced diameter, red blood cell velocity, and red blood cell flux) relative to young adult rats.

An update to the Monro-Kellie doctrine to reflect tissue compliance after severe ischemic and hemorrhagic stroke.

High intracranial pressure (ICP) can impede cerebral blood flow resulting in secondary injury or death following severe stroke. Compensatory mechanisms include reduced cerebral blood and cerebrospinal fluid volumes, but these often fail to prevent raised ICP. Serendipitous observations in intracerebral hemorrhage (ICH) suggest that neurons far removed from a hematoma may shrink as an ICP compliance mechanism. Here, we sought to critically test this observation. We tracked the timing of distal tissue shrinkage (e.g. CA1) after collagenase-induced striatal ICH in rat; cell volume and density alterations (42% volume reduction, 34% density increase; p < 0.0001) were highest day one post-stroke, and rebounded over a week across brain regions. Similar effects were seen in the filament model of middle cerebral artery occlusion (22% volume reduction, 22% density increase; p ≤ 0.007), but not with the Vannucci-Rice model of hypoxic-ischemic encephalopathy (2.5% volume increase, 14% density increase; p ≥ 0.05). Concerningly, this 'tissue compliance' appears to cause sub-lethal damage, as revealed by electron microscopy after ICH. Our data challenge the long-held assumption that 'healthy' brain tissue outside the injured area maintains its volume. Given the magnitude of these effects, we posit that 'tissue compliance' is an important mechanism invoked after severe strokes.

An increase in AMPK/e-NOS signaling and attenuation of MMP-9 may contribute to remote ischemic perconditioning associated neuroprotection in rat model of focal ischemia.

Remote ischemic perconditioning (RIPerC) results in collateral enhancement and a reduction in middle cerebral artery occlusion (MCAO) induced ischemia. RIPerC likely activates multiple metabolic protective mechanisms, including effects on matrix metalloproteinases (MMPs) and protein kinases. Here we explore if RIPerC improves neuroprotection and collateral flow by modifying the activities of MMP-9 and AMPK/e-NOS. Age matched adult male Sprague Dawley rats were subjected to MCAO followed one hour later by RIPerC (3 cycles of 15 min ischemia). Animals were euthanized 24 h post-MCAO. Haematoxylin and Eosin (H&E) staining 24 h post-MCAO revealed a significant (p < 0.02) reduction in the infarction volume in RIPerC treated animals (24.9 ± 5.4%) relative to MCAO controls (42.5 ± 4.2, %). TUNEL staining showed a 42.6% reduction in the apoptotic cells with RIPerC treatment (p < 0.01). Immunoblotting in congruence with RT-PCR and Zymography showed that RIPerC significantly reduced MMP-9 expression and activity in RIPerC + MCAO group compared to MCAO group (218.3 ± 19.1% vs. 148.9 ± 12.05% (p < 0.01). Immunoblotting revealed that RIPerC was associated with a significant 2.5-fold increase in activation of p-AMPK compared to the MCAO group (p < 0.01) which was also associated with a significant increase in the e-NOS activity (p < 0.01). RIPerC resulted in reduction of infarction volume, decreased apoptotic cell death and attenuated MMP-9 activity. This together with the increased activity of p-AMPK and increase in p-eNOS may, in part explain the neuroprotection and sustained increase in blood flow observed with RIPerC following acute stroke.