Inhibiting metabotropic glutamate receptor 5 after stroke restores brain function and connectivity.

Stroke results in local neural disconnection and brain-wide neuronal network dysfunction leading to neurological deficits. Beyond the hyper-acute phase of ischaemic stroke, there is no clinically-approved pharmacological treatment that alleviates sensorimotor impairments. Functional recovery after stroke involves the formation of new or alternative neuronal circuits including existing neural connections. The type-5 metabotropic glutamate receptor (mGluR5) has been shown to modulate brain plasticity and function and is a therapeutic target in neurological diseases outside of stroke. We investigated whether mGluR5 influences functional recovery and network reorganization rodent models of focal ischaemia. Using multiple behavioural tests, we observed that treatment with negative allosteric modulators (NAMs) of mGluR5 (MTEP, fenobam and AFQ056) for 12 days, starting 2 or 10 days after stroke, restored lost sensorimotor functions, without diminishing infarct size. Recovery was evident within hours after initiation of treatment and progressed over the subsequent 12 days. Recovery was prevented by activation of mGluR5 with the positive allosteric modulator VU0360172 and accelerated in mGluR5 knock-out mice compared with wild-type mice. After stroke, multisensory stimulation by enriched environments enhanced recovery, a result prevented by VU0360172, implying a role of mGluR5 in enriched environment-mediated recovery. Additionally, MTEP treatment in conjunction with enriched environment housing provided an additive recovery enhancement compared to either MTEP or enriched environment alone. Using optical intrinsic signal imaging, we observed brain-wide disruptions in resting-state functional connectivity after stroke that were prevented by mGluR5 inhibition in distinct areas of contralesional sensorimotor and bilateral visual cortices. The levels of mGluR5 protein in mice and in tissue samples of stroke patients were unchanged after stroke. We conclude that neuronal circuitry subserving sensorimotor function after stroke is depressed by a mGluR5-dependent maladaptive plasticity mechanism that can be restored by mGluR5 inhibition. Post-acute stroke treatment with mGluR5 NAMs combined with rehabilitative training may represent a novel post-acute stroke therapy.

Performing Enriched Environment Studies to Improve Functional Recovery.

Physical therapy and social interactions between the stroke patient and healthcare professionals or relatives facilitate the process of recovery and promote improvement of lost neurological function after stroke. These observations can be mimicked in an experimental setting by multimodal stimulation provided in the concept of enriched environment. The enriched environment is a housing condition combining social interactions and sensorimotor stimulation that improves lost neurological function without affecting the extent of brain damage after experimental stroke. This chapter provides a detailed protocol on how to perform enriched housing experiments including conceptual and technical considerations as a tool to investigate mechanisms of recovery after brain injury.

A Novel In Vivo Model for Multiplexed Analysis of Callosal Connections upon Cortical Damage.

Brain damage is the major cause of permanent disability and it is particularly relevant in the elderly. While most studies focused on the immediate phase of neuronal loss upon injury, much less is known about the process of axonal regeneration after damage. The development of new refined preclinical models to investigate neuronal regeneration and the recovery of brain tissue upon injury is a major unmet challenge. Here, we present a novel experimental paradigm in mice that entails the (i) tracing of cortico-callosal connections, (ii) a mechanical lesion of the motor cortex, (iii) the stereological and histological analysis of the damaged tissue, and (iv) the functional characterization of motor deficits. By combining conventional microscopy with semi-automated 3D reconstruction, this approach allows the analysis of fine subcellular structures, such as axonal terminals, with the tridimensional overview of the connectivity and tissue integrity around the lesioned area. Since this 3D reconstruction is performed in serial sections, multiple labeling can be performed by combining diverse histological markers. We provide an example of how this methodology can be used to study cellular interactions. Namely, we show the correlation between active microglial cells and the perineuronal nets that envelop parvalbumin interneurons. In conclusion, this novel experimental paradigm will contribute to a better understanding of the molecular and cellular interactions underpinning the process of cortical regeneration upon brain damage.

Plasticity-Enhancing Effects of Levodopa Treatment after Stroke.

Dopaminergic treatment in combination with rehabilitative training enhances long-term recovery after stroke. However, the underlying mechanisms on structural plasticity are unknown. Here, we show an increased dopaminergic innervation of the ischemic territory during the first week after stroke induced in Wistar rats subjected to transient occlusion of the middle cerebral artery (tMCAO) for 120 min. This response was also found in rats subjected to permanent focal ischemia induced by photothrombosis (PT) and mice subjected to PT or tMCAO. Dopaminergic branches were detected in the infarct core of mice and rats in both stroke models. In addition, the Nogo A pathway was significantly downregulated in rats treated with levodopa (LD) compared to vehicle-treated animals subjected to tMCAO. Specifically, the number of Nogo A positive oligodendrocytes as well as the levels of Nogo A and the Nogo A receptor were significantly downregulated in the peri-infarct area of LD-treated animals, while the number of Oligodendrocyte transcription factor 2 positive cells increased in this region after treatment. In addition, we observed lower protein levels of Growth Associated Protein 43 in the peri-infarct area compared to sham-operated animals without treatment effect. The results provide the first evidence of the plasticity-promoting actions of dopaminergic treatment following stroke.

Effect of Anti-inflammatory Treatment with AMD3100 and CX3CR1 Deficiency on GABAA Receptor Subunit and Expression of Glutamate Decarboxylase Isoforms After Stroke.

Following stroke, attenuation of detrimental inflammatory pathways might be a promising strategy to improve long-term outcome. In particular, cascades driven by pro-inflammatory chemokines interact with neurotransmitter systems such as the GABAergic system. This crosstalk might be of relevance for mechanisms of neuronal plasticity, however, detailed studies are lacking. The purpose of this study was to determine if treatment with 1,1'-[1,4-phenylenebis(methylene)]bis[1,4,8,11-tetraazacyclotetradecane] (AMD3100), an antagonist to the C-X-C chemokine receptor type 4 (CXCR4) and partial allosteric agonist to CXCR7 (AMD3100) alone or in combination with C-X3-C chemokine receptor type 1 (CX3CR1) deficiency, affect the expression of GABAA subunits and glutamate decarboxylase (GAD) isoforms. Heterozygous, CX3CR1-deficient mice and wild-type littermates were subjected to photothrombosis (PT). Treatment with AMD3100 (0.5 mg/kg twice daily i.p.) was administered starting from day 2 after induction of PT until day 14 after the insult. At this time point, GABAA receptor subunits (α3, ß3, δ), GAD65 and GAD67, and CXCR4 were analyzed from the peri-infarct tissue and homotypic brain regions of the contralateral hemisphere by quantitative real-time PCR and Western Blot. Fourteen days after PT, CX3CR1 deficiency resulted in a significant decrease of the three GABAA receptor subunits in both the lesioned and the contralateral hemisphere compared to sham-operated mice. Treatment with AMD3100 promoted the down-regulation of GABAA subunits and GAD67 in the ipsilateral peri-infarct area, while the ß3 subunit and the GAD isoforms were up-regulated in homotypic regions of the contralateral cortex. Changes in GABAA receptor subunits and GABA synthesis suggest that the CXCR4/7 and CX3CR1 signaling pathways are involved in the regulation of GABAergic neurotransmission in the post-ischemic brain.

Nucleolin as a potential biomarker for canine malignant neoplasia.

Human nucleolin (NCL) is a multifunctional protein that is involved in diverse pathological processes. Recent evidences have shown that NCL is markedly overexpressed on the surface of most human cancer cells when compared to normal cells, being overexpressed in several malignant cells. Based on the exposed, the purpose of this pilot study is to investigate the expression pattern of NCL in canine malignant neoplasia and control groups. NCL expression at both messenger RNA and protein levels in the subcellular fractions were respectively detected by RT-PCR and western blotting, allowing to infer the NCL positivity rate in canine neoplasia. The identity of NCL amplicons obtained by RT-PCR was confirmed by Sanger sequencing and found to correspond to Canis lupus familiaris. Using flow cytometry, the blood cells expressing NCL from canine neoplasms were also identified using several cell surface markers and their levels quantified. These results showed that NCL expressed in lymphocytes, monocytes and neutrophils in dogs with malignant neoplasia is higher (> 50%) when compared with the control group. We found an increased expression of surface and cytoplasmic NCL in canine malignant neoplasia group, while nuclear NCL is predominantly found in the control group. Overall, this study discloses and identifies for the first time the presence of NCL in canine blood.

Age, Sex Hormones, and Circadian Rhythm Regulate the Expression of Amyloid-Beta Scavengers at the Choroid Plexus.

Accumulation of amyloid-beta (Aß) in the brain is thought to derive from the impairment of Aß clearance mechanisms rather than from its overproduction, which consequently contributes to the development of Alzheimer's disease. The choroid plexus epithelial cells constitute an important clearance route for Aß, either by facilitating its transport from the cerebrospinal fluid to the blood, or by synthesizing and secreting various proteins involved in Aß degradation. Impaired choroid plexus synthesis, secretion, and transport of these Aß-metabolizing enzymes have been therefore associated with the disruption of Aß homeostasis and amyloid load. Factors such as aging, female gender, and circadian rhythm disturbances are related to the decline of choroid plexus functions that may be involved in the modulation of Aß-clearance mechanisms. In this study, we investigated the impact of age, sex hormones, and circadian rhythm on the expression of Aß scavengers such as apolipoprotein J, gelsolin, and transthyretin at the rat choroid plexus. Our results demonstrated that mRNA expression and both intracellular and secreted protein levels of the studied Aß scavengers are age-, sex-, and circadian-dependent. These data suggest that the Aß-degradation and clearance pathways at the choroid plexus, mediated by the presence of Aß scavengers, might be compromised as a consequence of aging and circadian disturbances. These are important findings that enhance the understanding of Aß-clearance-regulating mechanisms at the blood-cerebrospinal fluid barrier.

Correction: Transthyretin expression in the postischemic brain.

[This corrects the article DOI: 10.1371/journal.pone.0221555.].

Triiodothyronine modulates neuronal plasticity mechanisms to enhance functional outcome after stroke.

The development of new therapeutic approaches for stroke patients requires a detailed understanding of the mechanisms that enhance recovery of lost neurological functions. The efficacy to enhance homeostatic mechanisms during the first weeks after stroke will influence functional outcome. Thyroid hormones (TH) are essential regulators of neuronal plasticity, however, their role in recovery related mechanisms of neuronal plasticity after stroke remains unknown. This study addresses important findings of 3,5,3'-triiodo-L-thyronine (T3) in the regulation of homeostatic mechanisms that adjust excitability - inhibition ratio in the post-ischemic brain. This is valid during the first 2 weeks after experimental stroke induced by photothrombosis (PT) and in cultured neurons subjected to an in vitro model of acute cerebral ischemia. In the human post-stroke brain, we assessed the expression pattern of TH receptors (TR) protein levels, important for mediating T3 actions.Our results show that T3 modulates several plasticity mechanisms that may operate on different temporal and spatial scales as compensatory mechanisms to assure appropriate synaptic neurotransmission. We have shown in vivo that long-term administration of T3 after PT significantly (1) enhances lost sensorimotor function; (2) increases levels of synaptotagmin 1&2 and levels of the post-synaptic GluR2 subunit in AMPA receptors in the peri-infarct area; (3) increases dendritic spine density in the peri-infarct and contralateral region and (4) decreases tonic GABAergic signaling in the peri-infarct area by a reduced number of parvalbumin+ / c-fos+ neurons and glutamic acid decarboxylase 65/67 levels. In addition, we have shown that T3 modulates in vitro neuron membrane properties with the balance of inward glutamate ligand-gated channels currents and decreases synaptotagmin levels in conditions of deprived oxygen and glucose. Interestingly, we found increased levels of TRß1 in the infarct core of post-mortem human stroke patients, which mediate T3 actions. Summarizing, our data identify T3 as a potential key therapeutic agent to enhance recovery of lost neurological functions after ischemic stroke.

Thyroid Hormones in the Brain and Their Impact in Recovery Mechanisms After Stroke.

Thyroid hormones are of fundamental importance for brain development and essential factors to warrant brain functions throughout life. Their actions are mediated by binding to specific intracellular and membranous receptors regulating genomic and non-genomic mechanisms in neurons and populations of glial cells, respectively. Among others, mechanisms include the regulation of neuronal plasticity processes, stimulation of angiogenesis and neurogenesis as well modulating the dynamics of cytoskeletal elements and intracellular transport processes. These mechanisms overlap with those that have been identified to enhance recovery of lost neurological functions during the first weeks and months after ischemic stroke. Stimulation of thyroid hormone signaling in the postischemic brain might be a promising therapeutic strategy to foster endogenous mechanisms of repair. Several studies have pointed to a significant association between thyroid hormones and outcome after stroke. With this review, we will provide an overview on functions of thyroid hormones in the healthy brain and summarize their mechanisms of action in the developing and adult brain. Also, we compile the major thyroid-modulated molecular pathways in the pathophysiology of ischemic stroke that can enhance recovery, highlighting thyroid hormones as a potential target for therapeutic intervention.

Transthyretin expression in the postischemic brain.

The unknown role of the carrier protein transthyretin (TTR) in mechanisms of functional recovery in the postischemic brain prompted us to study its expression following experimental stroke. Male C57/B6 mice (age 9 to 10 weeks) were subjected to permanent focal ischemia induced by photothrombosis (PT) and brain tissues were analyzed for ttr expression and TTR levels at 24 hours, 48 hours, 7 days and 14 days following the insult by RT-PCR, Western blot and immunohistochemistry. Fourteen days after PT, non-specific TTR-like immunoreactive globules were found in the ischemic core and surrounding peri-infarct region by immunohistochemistry that could not be allocated to DAPI positive cells. No TTR immunoreactivity was found when stainings were performed with markers for neurons (Neuronal Nuclei, NeuN), reactive astrocytes (glial fibrillary acidic protein, GFAP) or microglia (cluster of differentiation 68, CD68). In addition, we could not find TTR by immunoblotting in protein extracts obtained from the ischemic territory nor ttr expression by RT-PCR at all time points following PT. In all experiments, ttr expression in the choroid plexus and TTR in the mouse serum served as positive controls and recombinant legumain peptide as negative control. Together, our results indicate that TTR is not synthesized in brain resident cells in the ischemic infarct core and adjacent peri-infarct area. Thus, it seems unlikely that in situ synthesized TTR is involved in mechanisms of tissue reorganization during the first 14 days following PT.

Choroid plexus is an additional source of melatonin in the brain.

The cerebrospinal fluid melatonin is released from the pineal gland, directly into the third ventricle, or produced de novo in the brain from extrapineal melatonin sources leading to a melatonin concentration gradient in the cerebrospinal fluid. Despite the interest on this topic, the brain areas capable of producing melatonin are not yet clear. Bearing this in mind, we hypothesized that the choroid plexus (CP) could be one of these melatonin sources. We analyzed and confirmed the presence of the four enzymes required for melatonin synthesis in rat CP and demonstrated that arylalkylamine N-acetyltransferase shows a circadian expression in female and male rat CP. Specifically, this enzyme colocalizes with mitochondria in rat CP epithelial cells, an organelle known to be involved in melatonin function and synthesis. Then, we demonstrated that melatonin is synthesized by porcine CP explants, although without a circadian pattern. In conclusion, our data show that the CP is a local source of melatonin to the central nervous system, probably contributing to its high levels in the cerebrospinal fluid. We believe that in the CP, melatonin might be regulated by its endogenous clock machinery and by the hormonal background.

The role of dopaminergic immune cell signalling in poststroke inflammation.

Upon ischaemic stroke, brain-resident and peripheral immune cells accumulate in the central nervous system (CNS). Interestingly, these cells express pattern specific to neurotransmitter receptors and, therefore, seem to be susceptible to neurotransmitter stimulation, potentially modulating their properties and functions. One of the principal neurotransmitters in the CNS, dopamine, is involved in the regulation of processes of brain development, motor control and higher brain functions. It is constantly released in the brain and there is experimental and clinical evidence that dopaminergic signalling is involved in recovery of lost neurological function after stroke. Independent studies have revealed specific but different patterns of dopamine receptor subtypes on different populations of immune cells. Those patterns are dependent on the activation status of cells. Generally, exposure to dopamine or dopamine receptor agonists decreases detrimental actions of immune cells. In contrast, a reduction of dopaminergic inputs perpetuates a pro-inflammatory state associated with increased release of pro-inflammatory molecules. In addition, subsets of immune cells have been identified to synthesize and release dopamine, suggesting autoregulatory mechanisms. Evidence supports that inflammatory processes activated following ischaemic stroke are modulated by dopaminergic signalling.

MicroRNA-124-loaded nanoparticles increase survival and neuronal differentiation of neural stem cells in vitro but do not contribute to stroke outcome in vivo.

There is a high quest for novel therapeutic strategies to enhance recovery after stroke. MicroRNA-124 (miR-124) has been described as neuroprotective and anti-inflammatory molecule. Moreover, miR-124 is a well described enhancer of adult neurogenesis that could offer potentially beneficial effects. Herein, we used miR-124-loaded nanoparticles (miR-124 NPs) to evaluate their therapeutic potential in an in vitro and in vivo model of stroke. For that, neuroprotective and neurogenic responses were assessed in an in vitro model of stroke. Here, we found that miR-124 NPs decreased cell death and improved neuronal differentiation of subventricular zone (SVZ) neural stem cell cultures after oxygen and glucose deprivation. In contrast, intravenous injection of miR-124 NPs immediately after permanent focal ischemia induced by photothrombosis (PT) did not provide a better neurological outcome. In addition, treatment did not affect the number of 5-bromo-2'-deoxyuridine (BrdU)- and doublecortin/BrdU- positive cells in the SVZ at the study endpoint of 14 days after PT. Likewise, the ischemic insult did not affect the numbers of neuronal progenitors in the SVZ. However, in PT mice miR-124 NPs were able to specifically augment interleukin-6 levels at day 2 post-stroke. Furthermore, we also showed that NPs reached the brain parenchyma and were internalized by brain resident cells. Although, promising in vitro data could not be verified in vivo as miR-124 NPs treatment did not improve functional outcome nor presented beneficial actions on neurogenesis or post-stroke inflammation, we showed that our NP formulation can be a safe alternative for drug delivery into the brain.

The choroid plexus harbors a circadian oscillator modulated by estrogens.

The suprachiasmatic nucleus (SCN) of the hypothalamus is considered the master circadian oscillator in mammals. However, extra-SCN structures in the brain also display daily rhythms. Recently, we have demonstrated that the choroid plexus (CP) expresses core clock genes that are subjected to circadian regulation in a sex-dependent manner. By using CP explants cultured from female knock-in mice carrying the Period-luciferase transgene, we show that CP exhibits endogenous circadian rhythms of PERIOD2::LUCIFERASE expression. Furthermore, we demonstrate that estrogen declines following ovariectomy modulates the daily rhythm expression of Bmal1, Per1 and Per2 in female rat CP, corroborating data obtained in experiments where rat CP epithelial cell (CPEC) cultures were incubated with 17ß-estradiol (E2). The molecular mechanism underlying these effects was also investigated, and we provide evidence that the estrogen receptor (ER) mediates the response of clock genes to E2. In conclusion, our study proves that the CP harbors a circadian oscillator that is modulated by estrogens and demonstrates that E2 regulation occurs through an estrogen-receptor-dependent mechanism.

The effects of whole green tea infusion on mouse urinary bladder chemical carcinogenesis.

OBJECTIVE(S): Green tea (GT) is one of the most popular beverages worldwide whose beneficial effects on health have been demonstrated. Recent studies suggest that GT may contribute to reduction of cancer risk and progression. The aim of this study was to evaluate the effects of whole GT on urinary bladder chemical carcinogenesis in male and female ICR mice. MATERIALS AND METHODS: The GT characterization was performed using spectrophotometric methods. Urinary bladder lesions were induced using N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) by gavage during 10 weeks and whole GT (0.5%) was provided ad libitum during 20 weeks. RESULTS: Animals from groups BBN+GT and BBN only developed preneoplastic lesions. CONCLUSION: We did not observe any effects by GT infusion administration on urinary bladder cancer development.

A liver schwannoma observed in a female Sprague-Dawley rat treated with MNU.

BACKGROUND: Schwannoma is a tumor of the nervous system composed by Schwann cells. It can occur naturally in several tissues of the body in both humans and animals. Diaphragmatic hernia can be congenital or acquired and is defined as a protrusion of abdominal viscera into the thoracic cavity. MATERIALS AND METHODS: The animal was a female rat from an experiment of mammary tumor chemically induced. It was injected with N-methyl-N-nitrosourea (MNU) and died spontaneously at 22 weeks of age. RESULTS: The animal had a diaphragmatic hernia and a hemorrhagic and multicystic mass in the liver herniated lobule. Microscopically the liver displayed a well circumscribed mass that was a tumor with hemorrhagic areas, necrosis and Antoni A and Antoni B patterns. It also displayed occasional positivity to vimentin and diffuse positivity to S-100 and NSE. CONCLUSION: The tumor was a schwannoma with the origin in the Glisson's capsule.