Long-Term Alterations in Motor Skills, Neurogenesis and Astrocyte Numbers following Transient Cerebral Ischemia in Mice.

Background and Objectives. Neurogenesis is an integral process in post-stroke recovery, involving the recruitment of proliferating neuroblasts from neurogenic niches of the mammal brain. However, the role of neurogenesis in the long-term restoration following ischemic stroke is fragmented. Post-stroke motor dysfunction includes challenges in the proper, coordinated use of hands and is present in roughly two-thirds of human patients. In this study, we investigated chronic behavioral and biochemical alterations after transient cerebral ischemia in adult male mice. Materials and Methods: Twelve-week-old C57BL/6N male mice were used, and fMCAo lasting 60 min was induced. At multiple timepoints after fMCAo induction, a single pellet reaching task was performed. Six months after the procedure, we immunohistochemically determined the number of proliferating neuroblasts (BrdU and DCX-positive) and the number of differentiated astrocytes (GFAP-positive) in both brain hemispheres. Results: The reaching ability of fMCAo mice was impaired from one month to six months after the induction of ischemia. Neuroblast proliferation was increased in the ipsilateral SVZ, whereas GFAP+ cell count was elevated in the hippocampal DG of both hemispheres of the fMCAo group mice. Conclusions: Our current report demonstrates the long-term effects of transient cerebral ischemia on mice functional parameters and neurogenesis progression. Our data demonstrate that transient cerebral ischemia promotes a long-lasting regenerative response in the ipsilateral brain hemisphere, specifically in the neurogenic SVZ and DG regions.

Focal Cerebral Ischemia Induces Global Subacute Changes in the Number of Neuroblasts and Neurons and the Angiogenic Factor Density in Mice.

Background and Objectives: Dissecting the complex pathological cascade of an ischemic stroke in preclinical models is highly warranted to understand the course of this disease in humans. Neurogenesis and angiogenesis are integral for post-stroke recovery, yet it is not clear how these processes are altered months after an ischemic stroke. In this study, we investigated the changes that take place subacutely after focal cerebral ischemia in experimental adult male mice. Materials and Methods: Male 12-week-old C57BL/6 mice underwent a 60 min long fMCAo or sham surgery. Two months after the procedure, we examined the immunohistochemistry to assess the changes in neuroblast (DCX) and differentiated neuron (NeuN) numbers, as well as the density of the pro-angiogenic factor VEGF. Results: We found decreased neuroblast numbers in both brain hemispheres of the fMCAo mice: by more than 85% in the dentate gyrus and by more than 70% in the subventricular zone. No neuroblasts were found in the contralateral hemisphere of the fMCAO mice or the sham controls, but a small population was detected in the ipsilateral ischemic core of the fMCAo mice. Intriguingly, the number of differentiated neurons in the ipsilateral ischemic core was lower by 20% compared to the contralateral hemisphere. VEGF expression was diminished in both brain hemispheres of the fMCAo mice. Conclusions: Our current report shows that focal cerebral ischemia induces changes in neuroblast numbers and the pro-angiogenic factor VEGF in both cerebral hemispheres 2 months after an fMCAo in mice. Our data show that focal cerebral ischemia induces a long-term regenerative response in both brain hemispheres.

Time-Dependent Memory and Gait Improvement by Intranasally-Administered Extracellular Vesicles in Parkinson's Disease Model Rats.

We have recently demonstrated that extracellular vesicles (EVs) derived from the human teeth stem cells improve motor symptoms and normalize tyrosine hydroxylase (TH) expression in the nigrostriatal structures of Parkinson's disease (PD) model rats obtained by 6-hydroxydopamine (6-OHDA) unilateral injection into the medial forebrain bundle (MFB). The aim of this study was to clarify: (1) how long therapeutic effects persist after discontinuation of 17-day intranasal administration of EVs in 6-OHDA rats; (2) may EVs reverse cognitive (learning/memory) dysfunction in these PD model rats; (3) whether and how the behavioral improvement may be related to the expression of TH and Nissl bodies count in the nigrostriatal structures. Our results demonstrated that in 6-OHDA rats, gait was normalized even ten days after discontinuation of EVs administration. EVs successfully reversed 6-OHDA-induced impairment in spatial learning/memory performance; however, the beneficial effect was shorter (up to post-treatment day 6) than that revealed for gait improvement. The shorter effect of EVs coincided with both full normalization of TH expression and Nissl bodies count in the nigrostriatal structures, while slight but significant increase in the 6-OHDA-decreased Nissl count persisted in the substantia nigra even on the post-treatment day 20, supposedly due to the continuation of protein synthesis in the living cells. The obtained data indicate the usefulness of further studies to find the optimal administration regimen which could be translated into clinical trials on PD patients, as well as to clarify other-apart from dopaminergic-neuromodulatory pathways involved in the EVs mechanism of action.

GABA-containing compound gammapyrone protects against brain impairments in Alzheimer's disease model male rats and prevents mitochondrial dysfunction in cell culture.

Neuroinflammation, oxidative stress, decreased glucose/energy metabolism, and disrupted neurotransmission are changes that occur early in sporadic Alzheimer's disease (AD), manifesting as mild cognitive impairment. Recently, the imbalanced function of the gamma-aminobutyric acid (GABA) system was identified as a critical factor in AD progression. Thus, maintaining balance among neurotransmitter systems, particularly the GABA system, can be considered a beneficial strategy to slow AD progression. The present study investigated the effects of the compound gammapyrone, a molecule containing three GABA moieties: "free" moiety attached to the position 4 of the 1,4-dihydropyridine (DHP) ring, and two "crypto" moieties as part of the DHP scaffold. The "free" and "crypto" GABA moieties are linked by a peptide bond (-CONH-), resulting in a peptide-mimicking structure. In a nontransgenic male rat AD model generated by intracerebroventricular (icv) streptozocin (STZ) administration, gammapyrone (0.1 and 0.5 mg/kg ip) mitigated the impairment of spatial learning and memory, prevented astroglial and microglial neuroinflammation, and normalized acetylcholine breakdown and GABA biosynthesis. In PC12 cells, gammapyrone protected against oxidative stress, mitochondrial dysfunction and apoptosis caused by the mitochondrial toxin di-2-ethylhexyl phthalate (DEHP). Gammapyrone did not bind to GABA-A and GABA-B receptors in vitro; therefore, we cannot attribute its neuroprotective action to a specific interaction with GABA receptors. Nevertheless, we suggest that the peptide-like regulatory mechanisms of gammapyrone or its allosteric modulatory properties are essential for the observed effects. Since, the icv STZ model resembles the early stages of AD, gammapyrone, and/or its congeners could be useful in the design of anti-dementia drugs.

Long-term behavioural alterations in mice following transient cerebral ischemia.

Ischemic stroke is one of the leading causes of disability and mortality worldwide. Acute and chronic post-stroke changes have variable effects on the functional outcomes of the disease. Therefore, it is imperative to identify what daily activities are altered after stroke and to what extent, keeping in mind that ischemic stroke patients often have long-term post-stroke complications. Translational studies in stroke have also been challenging due to inconsistent study design of animal experiments. The objective of this study was to clarify whether and to what extent mouse behaviour was altered during a 6 months period after cerebral stroke. Experimental stroke was induced in mice by intraluminal filament insertion into the middle cerebral artery (fMCAo). Neurological deficits, recovery rate, motor performance, and circadian activity were evaluated following ischemia. We observed severe neurological deficits, motor impairments, and delay in the recovery rate of mice during the first 14 days after fMCAo. Aberrant circadian activity and distorted space map were seen in fMCAo mice starting one month after ischemia, similarly to altered new and familiar cage activity and sucrose preference using the IntelliCage, and was still evident 60- and 180- days following stroke in the voluntary running wheel using the PhenoMaster system. A preference towards ipsilateral side turns was observed in fMCAo mice both acutely and chronically after the stroke induction. Overall, our study shows the importance of determining time-dependent differences in the long-term post-stroke recovery (over 180 days after fMCAo) using multiple behavioural assessments.

Neuroprotective potential of antihyperglycemic drug metformin in streptozocin-induced rat model of sporadic Alzheimer's disease.

The earliest hallmarks of sporadic Alzheimer's disease (sAD) are impaired glucose metabolism, chronic neuroinflammation, diminished synaptic plasticity and subsequent cognitive decline. The safest antidiabetic drug metformin has shown both glucose metabolism-improving and cognition-enhancing action in type 2 diabetes patients and diabetic model animals. However, metformin has not been previously studied in intracerebroventricular streptozocin (STZ)-induced model of sAD. Therefore, our aim was to assess the preventive action of metformin in sAD model-rats. Firstly, the actions of metformin (75 and 100 mg/kg) on cognitive functions and sociability were examined. Secondly, we wanted to identify whether behavioral effects of metformin were provided via its action on brain glucose transport, neuronal/glial uptake and metabolism. Thirdly, the effects of metformin on neuroinflammation, acetylcholine esterase density and activity, as well as on synaptic plasticity were determined. Our results showed that metformin reversed STZ-induced impairments in spatial learning/memory performance and sociability, coinciding with normalization of brain glucose transport, uptake and metabolism. Microgliosis and astrogliosis were ameliorated by metformin in sAD model rats. Metformin also preserved hippocampal synaptic plasticity and normalized acetylcholine cleavage in the cortical and hippocampal tissues, as well as inhibited acetylcholine esterase activity in vitro. These data indicate the promise of further research of metformin in early brain pathologies to stop neurodegenerative before severe cognitive decline occurs.

Neuroprotective action of diazepam at very low and moderate doses in Alzheimer's disease model rats.

Early manifestations of Alzheimer's disease (AD) include neuroinflammation, disrupted neurotransmission and cognitive deficits. Impairment of the GABAergic system is essentially involved in the pathogenesis of AD. Traditionally, agonists of GABAA receptors at doses above 1 mg/kg are known to possess memory impairing effects. However, we have previously found that GABAA receptor GABA site ligand muscimol at very low doses acted contrary - enhanced spatial learning/memory, as well as prevented neuroinflammation and augmented neurotransmission in AD model rats. Therefore, in the present study we focused on the assessment of the effects of non-sedative - very low (0.05 mg/kg) and moderate (1 mg/kg) - doses of diazepam, a positive allosteric modulator of benzodiazepine site of GABAA receptors. Its effects on spatial learning/memory and brain proteins related to neuroinflammation (GFAP and Iba-1), synaptic plasticity (SYP1), as well as acetylcholine breakdown and GABA biosynthesis were studied. Non-transgenic AD model rats (intracerebroventricular streptozocin injection) were used with the aim to mimic the pre-dementia stage of AD in humans. The obtained data showed that diazepam at both doses protected against streptozocin induced detrimental effects by enhancing spatial learning/memory, preventing neuroinflammation, preserving synaptic plasticity, as well as normalizing the hippocampal and cortical protein expression related to acetylcholine breakdown and GABA biosynthesis. One may suggest that at low and moderate doses diazepam is targeting non-specific, probably allosteric GABAA receptor sites, thus leading to stimulatory effects that can be beneficial for diazepam use in early pre-dementia stages of AD.

Intranasal Administration of Extracellular Vesicles Derived from Human Teeth Stem Cells Improves Motor Symptoms and Normalizes Tyrosine Hydroxylase Expression in the Substantia Nigra and Striatum of the 6-Hydroxydopamine-Treated Rats.

Parkinson's disease (PD) is the second most common neurodegenerative disorder affecting millions of people worldwide. At present, there is no effective cure for PD; treatments are symptomatic and do not halt progression of neurodegeneration. Extracellular vesicles (EVs) can cross the blood-brain barrier and represent promising alternative to the classical treatment strategies. In the present study, we examined therapeutic effects of intranasal administration of EVs derived from human exfoliated deciduous teeth stem cells (SHEDs) on unilateral 6-hydroxydopamine (6-OHDA) medial forebrain bundle (MFB) rat model of PD. CatWalk gait tests revealed that EVs effectively suppressed 6-OHDA-induced gait impairments. All tested gait parameters (stand, stride length, step cycle, and duty cycle) were significantly improved in EV-treated animals when compared with 6-OHDA-lesion group rats. Furthermore, EVs slowed down numbers of 6-OHDA-induced contralateral rotations in apomorphine test. Improvements in motor function correlated with normalization of tyrosine hydroxylase expression in the striatum and substantia nigra. In conclusion, we demonstrated, for the first time, the therapeutic efficacy of intranasal administration of EVs derived from SHEDs in a rat model of PD induced by 6-OHDA intra-MFB lesion. Our findings could be potentially exploited for the development of new treatment strategies against PD.

Very low doses of muscimol and baclofen ameliorate cognitive deficits and regulate protein expression in the brain of a rat model of streptozocin-induced Alzheimer's disease.

Recent studies devoted to neuroprotection have focused on the role of the gamma-aminobutyric acid (GABA) system in regulating neuroinflammatory processes which play a key role in the neurodegenerative processes observed in Alzheimer's disease (AD) by inducing glial cell overactivation and impairing neurotransmission. Data on the efficacy of classical GABA-A and GABA-B receptor agonists (muscimol and baclofen, respectively) in animal models of AD are not available. Moreover, no published studies have examined the ability of optimal doses of these compounds to prevent neuroinflammation, the alterations in neurotransmission and cognitive deficits. In the present study, we used a non-transgenic rat model of AD obtained by intracerebroventricular streptozocin (STZ) injection and assessed the effects of muscimol and baclofen at very low doses (0.01-0.05mg/kg) on spatial memory and the expression of cortical and hippocampal proteins related to neuroinflammation, namely proteins involved in astroglial functions (glial fibrillary acidic protein, GFAP), GABA synthesis (GABA synthesizing enzyme, glutamic acid decarboxylase 67, GAD67) and acetylcholine degradation (acetylcholine esterase). The presented study demonstrated that in a rat model of STZ-induced AD both muscimol and baclofen at the tested doses exerted memory-enhancing and anti-inflammatory effects, as well as normalization of acetylcholine esterase and GABA expression. We suggested that the function of very low doses of GABA receptor agonists differs from typical GABA-related inhibition and may be mediated by the allosteric sites of GABA receptors or other non-specific cell regulatory pathways.