Microglial activation and tau propagate jointly across Braak stages

Great research but how EXACTLY do we prevent that from happening? Interested parties like stroke survivors want to know. WHOM do we talk to ensure such further research is done? I want specific names otherwise NOTHING WILL OCCUR!

Microglial activation and tau propagate jointly across Braak stages

• Tharick A. Pascoal,
• Andrea L. Benedet,
• Nicholas J. Ashton,
• Min Su Kang,
• Joseph Therriault,
• Mira Chamoun,
• Melissa Savard,
• Firoza Z. Lussier,
• Cécile Tissot,
• Thomas K. Karikari,
• Julie Ottoy,
• Sulantha Mathotaarachchi,
• Jenna Stevenson,
• Gassan Massarweh,
• Michael Schöll,
• Mony J. de Leon,
• Jean-Paul Soucy,
• Paul Edison,
• Kaj Blennow,
• Henrik Zetterberg,
• Serge Gauthier &
• Pedro Rosa-Neto 

Nature Medicine (2021)Cite this article

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Abstract

Compelling experimental evidence suggests that microglial activation is involved in the spread of tau tangles over the neocortex in Alzheimer’s disease (AD). We tested the hypothesis that the spatial propagation of microglial activation and tau accumulation colocalize in a Braak-like pattern in the living human brain. We studied 130 individuals across the aging and AD clinical spectrum with positron emission tomography brain imaging for microglial activation ([¹¹C]PBR28), amyloid-β (Aβ) ([¹⁸F]AZD4694) and tau ([¹⁸F]MK-6240) pathologies. We further assessed microglial triggering receptor expressed on myeloid cells 2 (TREM2) cerebrospinal fluid (CSF) concentrations and brain gene expression patterns. We found that [¹¹C]PBR28 correlated with CSF soluble TREM2 and showed regional distribution resembling TREM2 gene expression. Network analysis revealed that microglial activation and tau correlated hierarchically with each other following Braak-like stages. Regression analysis revealed that the longitudinal tau propagation pathways depended on the baseline microglia network rather than the tau network circuits. The co-occurrence of Aβ, tau and microglia abnormalities was the strongest predictor of cognitive impairment in our study population. Our findings support a model where an interaction between Aβ and activated microglia sets the pace for tau spread across Braak stages.

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Cognitive Recovery After Stroke: A Meta-analysis and Metaregression of Intervention and Cohort Studies

 NOTHING HERE suggests that this will recover your 5 lost years of brain cognition from your stroke! So our researchers have a lot of work yet to do. WHOM will lead that effort? Specific names only. Decent leaders would take on this challenge and solve it, but we have none.  Your children and grandchildren will be screwed when they have strokes

Cognitive Recovery After Stroke: A Meta-analysis and Metaregression of Intervention and Cohort Studies

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Juan Pablo Saa, PhD, MPH, OTD, Tamara Tse, PhD, Carolyn M. Baum, PhD,

First Published May 22, 2021 Review Article 

https://doi.org/10.1177/15459683211017501

Article information

Abstract

Background

Cognition affects poststroke recovery, but meta-analyses of cognition have not yet provided a comparison of observational and intervention evidence.

Objective

To describe the trajectory of poststroke cognition and the factors that moderate it across intervention and observational cohorts.

Methods

Six databases were searched up to January 2020. Studies describing quantitative changes in cognition in adults poststroke were included. Interventions were classified into pharmacological, therapist-led, nonroutine/alternative, and usual care. Summary estimates were compared via hierarchical mixed-effects models. Age, recovery stage, stroke etiology, cognitive domain targeted in studies, and intervention types were investigated as moderators of cognition. Recovery stage and intervention were further analyzed in a multiplicative metaregression model.

Results

A total of 43 intervention trials and 79 observation cohorts involving 28 222 stroke participants were included. Heterogeneity was significant (τ² = 0.09; CI = 0.01-0.21, P < .001) with no evidence of publication bias. Cognitive recovery was greater in intervention trials (g = 0.47; CI = 0.37-0.58) than observational cohorts (g = 0.28; CI = 0.20-0.36) across all moderators analyzed. Nonroutine/alternative and pharmacological trials achieved the best overall results (g = 0.57, CI = 0.42-0.73, and g = 0.52, CI = 0.30-0.74, respectively), followed by therapist-led (g = 0.46; CI = 0.17-0.74), and usual care (g = 0.28; CI = 0.11-0.45) interventions. Medium recovery effects (ie, g ≥ 0.5) were observed in examining first-ever stroke, executive function, visuo-perceptual, consciousness, and psychomotor skills, 61 to 180 days poststroke, in participants aged 65 to 70 years.

Conclusion

Cognitive recovery is possible(NOT GOOD ENOUGH! Survivors do want recovery if you ever talk to them.) using different controlled interventions in all recovery stages, with smaller benefits ≥2 years poststroke. Longer-term studies are needed to determine the role of nonroutine/alternative therapies and the association between cognitive recovery and performance in everyday activities.

Keywords cognition, executive function, stroke, rehabilitation, meta-analysis, longitudinal studies

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Epicortical Brevetoxin Treatment Promotes Neural Repair and Functional Recovery after Ischemic Stroke

Now we just need followup research in humans. WHOM IS GOING TO DO THAT? Specific names only.

Epicortical Brevetoxin Treatment Promotes Neural Repair and Functional Recovery after Ischemic Stroke 

by Erica Sequeira ¹, Marsha L. Pierce ¹, Dina Akasheh ¹, Stacey Sellers ¹, William H. Gerwick ², Daniel G. Baden ³ and Thomas F. Murray ^1,*

¹

Department of Pharmacology and Neuroscience, Creighton University, Omaha, NE 68123, USA

²

Center for Marine Biotechnology & Biomedicine, Scripps Institution of Oceanography, San Diego, La Jolla, CA 92093, USA

³

Center for Marine Science University of North Carolina Wilmington, Wilmington, NC 28409, USA

^*

Author to whom correspondence should be addressed.

Mar. Drugs 2020, 18(7), 374; https://doi.org/10.3390/md18070374

Received: 25 June 2020 / Revised: 17 July 2020 / Accepted: 17 July 2020 / Published: 21 July 2020

(This article belongs to the Special Issue Marine Natural Products against Brain Diseases and Injuries)

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Abstract

Emerging literature suggests that after a stroke, the peri-infarct region exhibits dynamic changes in excitability. In rodent stroke models, treatments that enhance excitability in the peri-infarct cerebral cortex promote motor recovery. This increase in cortical excitability and plasticity is opposed by increases in tonic GABAergic inhibition in the peri-infarct zone beginning three days after a stroke in a mouse model. Maintenance of a favorable excitatory–inhibitory balance promoting cerebrocortical excitability could potentially improve recovery. Brevetoxin-2 (PbTx-2) is a voltage-gated sodium channel (VGSC) gating modifier that increases intracellular sodium ([Na⁺]i), upregulates N-methyl-D-aspartate receptor (NMDAR) channel activity and engages downstream calcium (Ca²⁺) signaling pathways. In immature cerebrocortical neurons, PbTx-2 promoted neuronal structural plasticity by increasing neurite outgrowth, dendritogenesis and synaptogenesis. We hypothesized that PbTx-2 may promote excitability and structural remodeling in the peri-infarct region, leading to improved functional outcomes following a stroke. We tested this hypothesis using epicortical application of PbTx-2 after a photothrombotic stroke in mice. We show that PbTx-2 enhanced the dendritic arborization and synapse density of cortical layer V pyramidal neurons in the peri-infarct cortex. PbTx-2 also produced a robust improvement of motor recovery. These results suggest a novel pharmacologic approach to mimic activity-dependent recovery from stroke.

Keywords: brevetoxin; ischemic stroke; peri-infarct; neuroplasticity

1. Introduction

Ischemic stroke is a common neurological disorder and major cause of long-term disability worldwide [1]. Currently, tissue plasminogen activator (tPA) is the only FDA-approved pharmacologic treatment for ischemic or thrombotic stroke, which carries the risk of producing an intracerebral hemorrhage [2,3,4]. Although this pharmacologic advancement of acute care has resulted in a decline in mortality rate, it has produced a greater number of disabled survivors. Soon after stroke onset, oxygen-deprived neurons in the infarct core cease to function while tissue in the surrounding peri-infarct region remain viable but compromised [5]. Previous work has suggested parallels between plasticity mechanisms in the developing brain and those occurring in the adult brain after a stroke event [6,7,8,9,10]. Neuronal circuits do undergo limited re-mapping and reorganization after stroke, and these repair processes are associated with neurogenesis, dentritogenesis, synaptogenesis, axonal sprouting and rewiring of cortical networks in the peri-infarct tissue [11]. However, this spontaneous reorganization only partially restores motor function. To more fully regain recovery of motor function, additional pharmacologic manipulations in the peri-infarct are required.

Glutamate-mediated excitotoxicity has been shown to contribute to ischemic cell death due to failure of ionic homeostasis and a sustained elevation of intracellular calcium concentration [12]. Following the excitotoxicity-induced acute insult, there is a period of recovery with characteristic heightened neuroplasticity in the peri-infarct tissue [13]. It is therefore critical that pharmacologic treatments to promote recovery be administered subsequent to the acute phase of the stroke. In rodent stoke models, pharmacologic and genetic strategies that enhance neuronal excitability in the peri-infarct cortex adjacent to the stroke promote motor recovery [14]. These mechanisms that enhance neuronal plasticity are similar to those involved in learning and memory [13]. In this regard, it is noteworthy that N-methyl-D-aspartate ionotropic glutamate receptors (NMDARs) are crucial in activity-dependent synaptic changes and in learning and memory.

Previous research has shown that changes in intracellular sodium concentration ([Na⁺]_i) produced in the soma and dendrites as a result of neuronal activity may act as a signaling molecule and play a role in activity-dependent synaptic plasticity. Synaptic stimulation elevates [Na⁺]_i to 10 mm in dendrites and up to 35–40 mm in dendritic spines [15]. In hippocampal neurons, such intracellular [Na⁺] increments have been demonstrated to increase NMDAR-mediated whole-cell currents and NMDAR single-channel activity by increasing both channel open probability and mean open time [16].

Brevetoxins (PbTx-1 to PbTx-10) are potent lipid soluble polyether neurotoxins produced by the marine dinoflagellate Karenia brevis [17]. PbTx-2 interacts with neurotoxin site 5 on the α subunit of voltage-gated sodium channels (VGSCs), and augments sodium influx by inhibiting channel inactivation and shifting the activation potential to more negative values [18]. Src kinases are widely expressed in the brain and regulate activities of ion channels such as NMDARs. Phosphorylation of NMDAR tyrosine residues by Src facilitates the binding of Na⁺ to NMDAR and exerts a regulatory effect on NMDAR signaling [19]. Single-channel currents recorded from cell-attached patches on cerebrocortical neurons indicate that PbTx-2 upregulates NMDAR whole-cell currents by increasing mean open time and probability without affecting the resting membrane potential [20]. This upregulation is attributed to the coincident elevation of intracellular [Na⁺] and Src kinase activation [21]. PbTx-2 treatment of cerebrocortical neuron cultures robustly potentiated NMDAR-mediated calcium influx (Ca²⁺) [20]. In immature cerebrocortical neurons, PbTx-2 treatment enhanced neurite outgrowth, dendritic arborization, synaptogenesis and filopodia formation and maturation [22]. In addition, PbTx-2 exposure engaged downstream activity-dependent mechanisms involved in neuronal growth and survival such as Ca²⁺-calmodulin kinases (CaMKs), extracellular signal-regulated kinase (ERK), cAMP response element binding protein (CREB) and brain-derived neurotrophic factor (BDNF) signaling pathways [22]. PbTx-2 exhibited a characteristic bidirectional concentration–response profile similar to that of NMDA since an optimal window for [Ca²⁺]_i is required for neurite extension and branching [23]. Inasmuch as the mechanisms involved in repair processes after stroke are similar to those regulating neuronal development, we hypothesized that PbTx-2 may augment recovery following ischemic stroke.

We therefore explored neurohistochemical and functional outcomes of administration of PbTx-2 during the recovery phase after stroke. To assess neurohistochemical changes, we imaged neurons in the peri-infarct cortex to assess dendritic arborization and synaptic density. In humans, long-term disabilities related to stroke often include impairments in feeding, coordination and gait. To examine impairment and recovery, we utilized a catwalk test to examine gross motor gait, a pasta matrix reach task to assess fine-motor skills (feeding and coordination) and a foot fault task to examine coordination and gait. An emerging strategy in stroke therapy is the direct application of treatments to the stroke lesion [24,25,26]. Accordingly, we mixed PbTx-2 in a hydrogel composed of thiol-modified hyaluronan and polyethylene glycol diacrylate and this composite was deposited epicortically directly above the stroke cavity. We demonstrate that epicortical application of PbTx-2 at five-days post-infarct enhances neuronal repair and improves functional outcomes.

2. Results

2.1. PbTx-2 Enhances Neuronal Structural Plasticity in the Peri-Infarct Region as Revealed by Increased Dendritic Arbor Complexity and Synapse Formation

Using 2- to 4-month-old male yellow fluorescent protein (YFP) line-H transgenic mice, we produced unilateral photothrombotic strokes by providing an intraperitoneal injection of a light sensitive dye followed by exposure of the motor cortex region to a cold source of light. Mice were then allowed to recover in home cages for five days (Figure 1). On day 5, animals were treated with the vehicle or PbTx-2 and subsequently sacrificed on day 6 to examine neuronal structural changes in the peri-infarct region.

Figure 1. Experimental timeline: Animals were food-restricted to 85% of their body weight for two weeks prior to training for pasta matrix reach task. Animals were trained to perform pasta matrix reach task and foot fault task prior to inducing stroke to obtain baseline values. A focal lesion was induced in the motor cortex region by photothrombosis on day 0. Animals were randomly divided into PbTx-2 or vehicle treatment groups. On day 5, PbTx-2 mice were treated with 3, 10, 100, 1000 or 3000 pmol PbTx-2, whereas vehicle-treated animals were given hydrogel alone. Further, on day 1 (post-stroke) and day 6 (post-treatment), animals were assessed for the number of pasta pieces retrieved and percentage of foot faults. On day 6, animals were sacrificed, and brains were isolated for histological analysis of surviving neurons in the peri-infarct region.

We performed cresyl violet staining to evaluate the extent of the photothrombotic stroke volume and the effect of epicortical application of PbTx-2 on the same (Figure 2A). Coronal sections of 100 μm were collected using a vibratome (Leica VT 1200S). For the infarct volume measurement, brain sections were stained with 0.5% cresyl violet and images were then acquired using a bright-field microscope. The areas of infarct were delineated and quantified using the Image J software (NIH) and infarct volume was calculated by summation of the lesion areas of all sections and integrated by the thickness of the section (Figure 2B). Infarct volumes did not vary significantly between vehicle- and PbTx-2-treated mice (Figure 2C). These results indicate that the timing of PbTx-2 treatment utilized was appropriate for the assessment of promoting functional recovery inasmuch as these treatments did not affect stroke volume.

Figure 2. Histologic assessments after stroke. (A) Representative cresyl violet image showing the infarct and the peri-infarct region. (B) Representative cresyl violet stained-sections of vehicle, 3, 10, 100, 1000 and 3000 pmol PbTx-2, respectively. (C) Quantification of infarct volume. No significant differences in infarct size were detected (one-way ANOVA followed by Dunnett’s post hoc test, 3 pmol PbTx-2 effect, p = 0.993; 10 pmol PbTx-2 effect, p = 0.907; 100 pmol PbTx-2 effect, p > 0.999; 1000 pmol PbTx-2 effect, p = 0.873; 3000 pmol PbTx-2 effect, p = 0.975). Data shown are mean ± SEM of 2 to 7 brains.

Dendritic injury is a pathologic hallmark of excitotoxicity inasmuch as NMDARs are predominantly localized on dendrites [27,28]. Stroke caused the deterioration of neurons in the infarct core that could be readily distinguished from the adjacent surviving peri-infarct region. Confocal images of layer V YFP-labelled pyramidal neurons in the peri-infarct region were obtained and 3D morphological analysis of the arbor complexity with a defined algorithm was performed using the Imaris image analysis software (Figure 3A). In vehicle-treated animals, there was a gradual increase in branching complexity moving away from the soma, reaching a maximum of 8 ± 0.5 intersections per neuron, and then progressively declining beginning at approximately 10 µm from the soma (Figure 3B). Doses of 10, 100 and 1000 pmols of PbTx-2 produced a 2-fold increase in the expansion of dendritic arbors of neurons in the peri-infarct cortex, and a rightward shift in the Sholl plot as compared with the vehicle-treated control mice (Figure 3B). An AUC analysis of Sholl data showed a significant increase in dendritic complexity following the 10, 100 and 1000 pmol doses of PbTx-2 compared with vehicle controls. The PbTx-2 effect on dendritic complexity displayed a bidirectional profile as shown by a lack of effect on the expansion of dendritic arbors at the 3 and 3000 pmol doses (one-way ANOVA followed by Dunnett’s post hoc test, **** p < 0.0001; 3 pmol PbTx-2 effect, p = 0.579; 3000 pmol PbTx-2 effect, p = 0.998; n = 17 to 51 neurons; Figure 3C). Given our previous demonstration of bidirectional PBTx-2 concentration–response profiles in cerebrocortical neurons similar to that of NMDA, a possible explanation for PbTx-2’s bidirectional profile herein is the underlying NMDAR-dependent mechanism of action [23]. Previous studies have established an inverted-U concentration–response profile for the relationship between NMDAR and neuronal survival, where too little or too large activation of NMDARs can respectively diminish neuronal growth or cause cell death [29].

Figure 3. Effect of PbTx-2 on dendritic arborization in the peri-infarct region. (A) Representative images of PbTx-2-induced dendritic arborization in the peri-infarct region (Scale bar: 30 μm). (B) Sholl analysis to quantify dendritic arbor complexity. (C) Area under the curve (AUC) analysis of Sholl data, PbTx-2 at 10, 100 and 1000 pmol doses enhanced dendritic arbor complexity in the peri-infarct site as compared with the vehicle-treated animals (one-way ANOVA followed by Dunnett’s post hoc test, **** p < 0.0001). However, the 3 and 3000 pmol doses of PbTx-2-treated mice were without effect on the expansion of dendritic arbors (3 pmol PbTx-2 effect, p = 0.579; 3000 pmol PbTx-2 effect, p = 0.998). Each bar represents the mean ± SEM of 17–51 neurons.

Next, we examined the influence of PbTx-2 on synapse formation in the peri-infarct cortex region. Confocal images of YFP-labelled neurites in the peri-infarct region were obtained and analyzed using a spot detection algorithm again using the Imaris image analysis software (Figure 4A). Antibodies against VGLUT1 (presynaptic marker) and PSD-95 (postsynaptic marker) were used to quantify synapse density, as revealed by colocalized fluorescent puncta (Figure 4B). We obtained the ratio of synaptic puncta on the ipsilesional side to that of the contralesional side to correct for between animal variation. Dose–response analysis of the effect of PbTx-2 on synapse density indicated that 3, 10, 100 and 1000 and 3000 pmol PbTx-2 produced a significant increase in synapse formation (number of puncta per length of neurite) compared with the vehicle-treated animals (one-way ANOVA followed by Dunnett’s post hoc test, **** p < 0.0001; n= 12 to18 neurons; Figure 4C).

Figure 4. Effect of PbTx-2 on excitatory synapse density in peri-infarct region. (A) Representative images of double-immunostained YFP expressing neurites in the peri-infarct region at day 6 obtained from a confocal microscope. (B) Antibodies against VGLUT1 (presynaptic marker/blue) and PSD-95/red (postsynaptic marker) were used to quantify synapse density, as indicated by colocalized fluorescent puncta (yellow), scale bar: 5 µm. (C) Quantification of colocalized fluorescent puncta using Imaris image analysis; 3, 10, 100, 1000 and 3000 pmol PbTx-2 doses enhanced synapse density in the peri-infarct region (one-way ANOVA followed by Dunnett’s post hoc test, **** p < 0.0001). (D) Gardner–Altman mean comparison plots showing PbTx-2-induced increments in synapse density. Each plot contains comparisons for all PbTx-2-treated groups, and each dot represents % ipsilateral/contralateral puncta from an individual brain section. (E) Gardner–Altman plot demonstrating effect size. The left ordinate axis of the plot shows mean difference (MD) distribution between the PbTx-2-treated and vehicle-treated groups; 3 pmol PbTx-2 MD = 37.6%, 95% CI [28.3, 53.4]; 10 pmol PbTx-2: MD = 63%, 95% CI [47.4, 86.2]; 100 pmol PbTx-2: MD = 52.9%, 95% CI [42.3, 60.7]; 1000pmol PbTx-2: MD = 51%, 95% CI [40.3, 60.6]; 3000 pmol PbTx-2: MD = 38.7%, 95% CI [32.4, 47.0 All data are represented as mean ± SEM of 12–18 brain sections.

Further, to better visualize the PbTx-2 effect size, we compared mean difference (MD) distribution between the vehicle- and PbTx-2-treated groups using the Gardner–Altman mean comparison plot that affords transparency of the effect size of treatments [30] (3 pmol PbTx-2 MD = 37.6%, 95% CI [28.3, 53.4]; 10 pmol PbTx-2: MD = 63%, 95% CI [47.4, 86.2]; 100 pmol PbTx-2: MD = 52.9%, 95% CI [42.3, 60.7]; 1000pmol PbTx-2: MD = 51%, 95% CI [40.3, 60.6]; 3000 pmol PbTx-2: MD = 38.7%, 95% CI [32.4, 47.0]; Figure 4D,E). The effect sizes and CIs are reported as: effect size [CI width—lower bound, upper bound] and the narrowness of the confidence interval represents the effect size precision.

2.2. PbTx-2 Promotes Recovery of Fine Motor Skills in Stroke Affected Mice

We next assessed the influence of the photothrombotic focal stroke on motor function and coordination by first performing a CatWalk gait analysis. We examined contralateral forelimb function post-stroke as motor disability in response to photothrombotic stroke [31]. We analyzed several gait parameters on days 1, 2 and 5 post-stroke and compared the stroke ipsilateral left front (LF) paw to the contralateral right front (RF) paw in each animal. No differences between the sham-operated group and photothrombotic stroke (PTS) group were detected. Shown are the results for parameters “max contact”, “intensity” and “stride length” (unpaired two-tailed test: max contact (%), p = 0.721; intensity (%), p = 0.987; stride length (%), p = 0.471; n = 9 mice; Figure 5). These results demonstrated a lack of effect of the stroke on gait parameters and that motor deficits might be confined to the digits of the paw. We therefore next used a pasta matrix reach task and a foot fault test to assess fine motor skills.

Figure 5. Impact of photothrombotic stroke on gross motor function. Representative CatWalk parameters (A) max contact (%), (B) intensity (%) and (C) stride length (%) analyzed before inducing photothrombotic stroke (PTS) to obtain baseline and on days 1, 2 and 5 after stroke. We compared the ipsilateral left front (LF) paw to the contralateral right front (RF) paw. Gait parameters were not altered by photothrombotic stroke. Shown are the results of unpaired two-tailed test (max contact (%), p = 0.721; intensity (%), p = 0.987; stride length (%), p = 0.471). All values are given as mean ± SEM (n = 9 mice).

Rodents live in an environment that requires the use of a complex range of motor skills to gain access to food [32]. The pasta matrix reach task is one of the few motor tests that can measure skilled forepaw use [33]. Mice were trained for three days to grab a single piece of pasta to determine paw preference (Figure 6A), followed by seven days of pasta matrix training sessions to obtain baseline values before inducing stroke (Figure 6B). On day 1, following the stroke, there was a significant decline in the number of pasta pieces retrieved, indicating that the photothrombotic stroke affected fine motor skills. On day 6, following the stroke, animals treated with the 10, 100 and 1000 pmol doses of PbTx-2 exhibited a significant increase in the number of pasta pieces retrieved as compared with the vehicle-treated mice. The lowest and highest doses of PbTx-2, 3 and 3000 pmol, were however without a significant effect (one-way ANOVA followed by Dunnett’s post hoc test, **** p < 0.0001, *** p = 0.0002; 3 pmol PbTx-2 effect, p = 0.955; 3000 pmol PbTx-2 effect, p = 0.873; n = 9 to 15 mice; Figure 6).

Figure 6. Effect of PbTx-2 treatment on pasta matrix reach task. (A) Paw preference was determined using a single piece of pasta for 3 days. (B) Animals were trained for 7 days to retrieve 25 pieces placed in a 5X5 matrix from their preferred paw. Day 0 represents the photothrombotic procedure and at 1 day after the stroke, there was a significant reduction in the number of pasta pieces retrieved, indicating motor deficits induced by the insult. On day 6, after the stroke, the 10, 100 and 1000pmol PbTx-2-treated animals exhibited significant increases in the number of pasta pieces retrieved as compared with the vehicle-treated animals. The 3 and 3000 pmol PbTx-2 doses did not facilitate functional recovery. (C) Quantification of PbTx-2 dose–response effects on motor recovery 6 days post-stroke (one-way ANOVA followed by Dunnett’s post hoc test, **** p < 0.0001, ***p = 0.0002; 3 pmol PbTx-2 effect, p = 0.955; 3000 pmol PbTx-2 effect, p = 0.873). All data are represented as mean ± SEM (n = 9 to 15 mice). Data points without error bars are due to the error bar being smaller than the symbol.

To further confirm the influence of PbTx-2 on functional recovery, we used a foot fault task that represents a sensitive method for detecting motor deficits of limb functioning and placement during locomotion. Animals without a stroke should place their paws precisely on the wire frame and demonstrate few to zero foot faults [33]. The photothrombotic stroke in the forelimb motor cortex produced a significant increase in the percentage foot faults on day 1 after the insult, indicating a disruption of limb function and placement (Figure 7A). At PbTx-2 doses of 10, 100 and 1000 pmols, treated animals displayed significant improvements in percentage of foot faults as compared with the vehicle-treated mice. Again, the 3 and 3000 pmol doses of PbTx-2 did not promote functional recovery (one-way ANOVA followed by Dunnett’s post hoc test, 10 pmol PbTx-2 effect; * p = 0.023, 100 pmol PbTx-2 effect; * p = 0.027, 1000 pmol PbTx-2 effect; ** p = 0.001; 3 pmol PbTx-2 effect, p = 0.703; 3000 pmol PbTx-2 effect, p > 0.999; n = 9 to 13 mice; Figure 7B). These data establish that treatment with the 10, 100 and 1000 pmol doses of PbTx-2 on day 5 post-stroke resulted in functional recovery of fine motor skills in both the pasta matrix handling and foot fault tasks as compared with the vehicle control treatments.

Figure 7. Effect of PbTx-2 treatment on foot fault task. (A) Animals were trained to walk on an elevated grid prior to photothrombotic stroke to obtain baseline values. On day 1 after inducing stroke, a significant increase in percentage of foot faults was observed. The 10, 100 and 1000 pmol doses of PbTx-2 produced significant improvement in percentage of foot faults as compared with the vehicle-treated animals. Alternatively, the 3 and 3000 pmol doses of PbTx-2 did not aid recovery. (B) Quantification of PbTx-2 dose–response effects on motor recovery 6 days post-stroke (one-way ANOVA followed by Dunnett’s post hoc test, 10 pmol PbTx-2 effect, * p = 0.023; 100 pmol PbTx-2 effect, * p = 0.027; 1000 pmol PbTx-2 effect, ** p = 0.001; 3 pmol PbTx-2 effect, p = 0.703; 3000 pmol PbTx-2 effect, p > 0.999). All data points are represented as mean ± SEM (n = 9 to 13 mice).

3. Discussion

Here, we investigated the effect of PbTx-2 on neuroplasticity in the peri-infarct cortex and associated motor functions in a murine model of stroke. The main findings of this study are: 1. epicortical application of PbTx-2 at the stroke site produced a 2-fold increase in dendritic arborization and increased synaptogenesis in the peri-infarct cortex, 2. photothrombotic stroke in the forelimb motor cortex produced functional deficits that were confined to the digits of the paw, 3. PbTx-2 doses of 10, 100 and 1000 pmols produced dramatic improvements in functional recovery toward pre-stroke controls as measured by an increase in the number of pasta pieces retrieved or decreased percentage of foot faults and 4. PbTx-2 displayed bidirectional dose–response profiles where the 3 and 3000 pmol doses did not affect neurite outgrowth or motor functional recovery, consistent with these effects being mediated through NMDARs.

VGSCs play a fundamental role in electrical signaling of the nervous system and action potential generation [34]. Two-photon imaging studies show that synaptic stimulation leads to transient increases in [Na⁺]_i in postsynaptic spines and dendrites [15]. This suggests that [Na⁺]_i may function as a signaling molecule and play a role in activity-dependent synaptic plasticity. PbTx-2, a VGSC gating modifier, augments NMDA receptor signaling through coincidence of an elevation of [Na⁺]_i and Src kinase activity [21]. A previous report suggested that PbTx-2-mediated activation of sodium channels was associated with enhancement of NMDA-induced Ca²⁺ influx, accelerated spine formation and maturation, increased dendritic arbor elaboration and increased synaptogenesis in developing cerebrocortical neurons [22]. The cell signaling mechanisms underlying these responses involved PbTx-2-induced increase in intracellular Ca²⁺ with attendant phosphorylation of Ca²⁺-dependent molecules including CaMKI, CaMKII and CREB that play essential roles in neuronal growth and survival. BDNF is an activity-dependent neurotrophic factor that mediates neuroplasticity and is regulated by CREB-dependent mechanisms [35], and PbTx-2 exposure also increased the surface expression of BDNF-tropomyosin-related kinase B receptors in cerebrocortical neurons [22].

Glutamate plays an essential role in mediating excitatory neurotransmission in the central nervous system and is vital for synaptic plasticity. After an ischemic stroke, however, glutamate accumulation leads to excitotoxicity due to over-activation of NMDARs and neuronal death [36,37]. Interestingly, NMDAR antagonists failed clinically to show neuroprotective effects and, in some cases, worsened stroke outcomes in patients [38,39,40,41]. Hence, blocking NMDARs subsequent to a stroke is detrimental inasmuch as glutamate signaling through NMDARs contributes to neuronal survival. This influence of glutamate on NMDARs to promote neuronal survival displays an inverted U-shaped concentration–response curve, where too little or excessive activation of NMDARs are detrimental [42].

Neuronal excitability after a stroke exhibits distinct phases during stroke progression and recovery [43]. In the acute phase, excessive glutamatergic activity produces excitotoxicity and is deleterious. During the subsequent chronic phase however, glutamatergic excitability in the peri-infarct cortex is correlated with neuronal repair and recovery [43]. Therefore, enhancing cortical excitability too early after stroke may further increase neuronal death. This inflection point from the acute excitotoxic to chronic recovery phase occurs three days post-stroke in mice [14]. In the present study, we therefore selected the time point of five days after stroke for the epicortical treatments. Stroke recovery has been associated with dramatic spine plasticity in the peri-infarct cortex and with an increase in dendritic spine density over baseline values in some regions [11]. This influence of glutamatergic signaling is opposed by a marked increase in extracellular γ-aminobutyric acid (GABA) levels due to the loss of GABA transporter GAT-3 [14]. Notably, administration of L655,708, a benzodiazepine inverse agonist specific for extrasynaptic GABA_A receptors, produced a rapid and sustained improvement in functional recovery in mice following a photothrombotic stroke [14]. Hence, counteracting the hypo-excitability caused by heightened GABAergic inhibition could potentially promote recovery when initiated during the chronic phase. The present results using the sodium channel gating modifier brevetoxin may provide an additional approach for enhancing brain excitability during the period of recovery and reorganization to promote neural repair.

We selected the photothrombotic stroke model because it produces a localized infarct that permits a detailed analysis of neuronal structural plasticity and functional recovery [44]. The effect of photothrombotic stroke on forelimb fine motor deficits, as assessed by the pasta matrix reach and foot fault tasks, appeared to be both pervasive and persistent since at six days post-stroke, the vehicle-treated animals displayed profound deficits in task performance. We found that a single epicortical PbTx-2 treatment applied five days post-stroke was sufficient to promote functional recovery and that these beneficial effects were paralleled by PbTx-2-induced increases in dendritic arbor complexity and synaptic density in the peri-infarct cortex. These actions of PbTx-2 on neuronal plasticity and functional recovery both showed inverted-U dose–response curves. We have shown previously that the in vitro effect of PbTx-2 on neurite outgrowth in cerebrocortical neurons exhibited a bidirectional concentration–response (inverted-U) profile and that this effect was primarily dependent on NMDARs [20]. Similarly, the effects of PbTx-2 on dendritic arborization and synaptogenesis in cerebrocortical neurons displayed bidirectional concentration–response profiles [22]. The inverted-U model for the relationship between NMDAR activity and neuronal survival and growth is well established [29]. The inverted-U dose–response effects of PbTx-2 on neuronal plasticity in the peri-infarct cortex observed in the present study are consonant with those of a previous report that neuronal activity affects structural plasticity in vivo through NMDAR-triggered intracellular signaling events [45]. It is therefore reasonable to posit that the effects of epicortical PbTx-2 on structural plasticity and post-stroke functional recovery are the result of elevated [Na⁺]_i and enhanced NMDAR function.

Our results demonstrate impairment in forelimb fine motor control in mice after a photothrombotic stroke and reversal of these deficits by PbTx-2 treatment. These data suggest that stroke-induced motor deficits might be particularly responsive to augmented cortical excitability during the recovery phase of stroke. Currently, the only clinical treatment following a stroke is tissue plasminogen activator (tPA) which must be administered within the first few hours post-stroke. Considering that occupational and physical therapy are the standard of care for stroke recovery, our results suggest that sodium channel gating modifiers may represent a novel pharmacotherapy to accelerate recovery. This new strategy to enhance cortical excitability during the delayed time frame important for neural repair and recovery may hold promise for reducing the severity of stroke disability.

4. Materials and Methods

Association of pre-stroke metformin use, stroke severity, and thrombolysis outcome

So that brings up an immediate question. 'Should metformin be immediately given to stroke patients as part of their hyperacute therapy?'  WHOM will answer that question? Specific names needed. You can see the fucking incompetence of all the stroke medical world in that nothing seems to have been done with this in the past 4.5 years.

You could stay forever young (or young for a long time) with this diabetes drug 

Dec. 2015

In this one is this line:The drug, which is cheaply available for just $0.16 a day, works by boosting the number of oxygen molecules released into a cell, which in turn seems to benefit the robustness and longevity of the body’s basic building blocks. (This would seem to be much easier and faster than HBOT. I'm requesting this at my next stroke, my doctor won't know what hit her when I tell her how to treat me.)

My list of 31 things I was going to demand after my next stroke. I guess metformin isn't in there.

 The latest here:

Association of pre-stroke metformin use, stroke severity, and thrombolysis outcome

Laura P Westphal, Roni Widmer, Ulrike Held, Klaus Steigmiller, View ORCID ProfileChristian Hametner, Peter Ringleb, Sami Curtze, Nicolas Martinez-Majander, Marjaana Tiainen, Christian H Nolte, Jan F Scheitz, Hebun Erdur, Alexandros A Polymeris, Christopher Traenka, Ashraf Eskandari, Patrik Michel, View ORCID ProfileMirjam R Heldner, Marcel Arnold, View ORCID ProfileAndrea Zini, Laura Vandelli, Jonathan M Coutinho, Adrien E Groot, Visnja Padjen, Dejana R Jovanovic, Yannick Bejot, Céline Brenière, Guillaume Turc, Pierre Seners, Alessandro Pezzini, Mauro Magoni, Didier Leys, Sixtine Gilliot, Michael J Scherrer, Georg Kägi, Andreas R Luft, Henrik Gensicke, Paul Nederkoorn, Turgut Tatlisumak, Stefan T Engelter, Susanne Wegener

First published June 29, 2020, DOI: https://doi.org/10.1212/WNL.0000000000009951

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Abstract

Objective: To evaluate whether pretreatment with metformin (MET) is associated with less stroke severity and better outcome after intravenous thrombolysis (IVT), we analyzed a cohort of 1919 stroke patients with type-2 diabetes in a multicenter exploratory analysis.

Methods: Data from patients with diabetes affected by ischemic stroke treated with IVT were collected within the European Thrombolysis in Ischemic Stroke Patients (TRISP) collaboration. We applied propensity score matching (PSM) to obtain balanced baseline characteristics of patients treated with and without MET.

Results: Of 1919 stroke patients with type-2 diabetes who underwent IVT, 757 (39%) had received MET before stroke (MET+), whereas 1162 (61%) had not (MET-). MET+ patients were younger with a male preponderance. Hypercholesterolemia and pretreatment with statins, antiplatelets or antihypertensives were more common in the MET+ group. After PSM, the two groups were well balanced with respect to demographic and clinical aspects. Stroke severity on admission (NIHSS 10.0 ± 6.7 vs. 11.3 ± 6.5), 3-months degree of independence on modified Rankin Scale (mRS): 2 [IQR 1.0, 4.0] vs. 3 [IQR 1.0, 4.0] as well as mortality (12.5% vs. 18%) were significantly lower in the MET+ group. The frequency of symptomatic intracerebral hemorrhages did not differ between groups. HbA1c levels were well balanced between both groups.

Conclusions: Stroke patients with diabetes on treatment with MET receiving IVT had less severe strokes on admission and a better functional outcome at 3 months. This suggests a protective effect of MET resulting in less severe strokes as well as beneficial thrombolysis outcome.

• Received April 19, 2019.
• Accepted in final form January 6, 2020.

• © 2020 American Academy of Neurology