The role of transthyretin and thyroid hormones on functional recovery after experimental stroke

Stroke remains one of the leading causes of death and disability worldwide. Focal ischemic cortical stroke results in tissue demise in the infarct core and neuronal dysfunction in areas surrounding the core. The loss of neuronal function triggers specific neuroanatomical and neurophysiological changes in both adjacent and remote areas during the first weeks after stroke onset. During this critical time window, there is a profound reorganization of cortical maps that is accompanied with spontaneous neuroplasticity, however, with limited and partially aberrant recovery of motor function. Induced plasticity with external interventions such as rehabilitation, facilitates recovery and promotes improvement of lost neurological function, albeit to a limited extent. Despite much effort has been spent in developing adjuvant therapies to foster spontaneous underlying endogenous mechanisms, none of the treatment attempts reached clinical use. Thus, rehabilitation remains the only evidence-based long-term treatment in stroke survivors. We hypothesized that by targeting thyroid hormones (TH) and their carrier protein transthyretin (TTR) might be a promising therapeutic strategy to foster endogenous mechanisms of neurorepair. TH are of fundamental importance for brain development and essential factors to warrant brain functions throughout life. In particular, the active form 3,5,3’-triiodo-L-thyronine (T3) is involved in the regulation of neuronal plasticity, stimulation of angiogenesis and neurogenesis as well as modulation of 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. Independent of its role as a TH carrier protein, TTR has been studied as a neuroprotective molecule in the brain, which has been emphasized as promising target to enhance lost neurological functions during the recovery phase after stroke. In the experimental setting, we investigated if TH and TTR are involved in the reorganization of cortical neuronal function after stroke. We found that administration of T3 50 μg/kg during the first two weeks after photothrombotic stroke in mice significantly enhanced functional recovery of lost neurological function without affecting infarct size. Motor improvement was accompanied by mechanisms of homeostatic regulation in the peri-infarct area in favor for an increased excitability. The mechanisms involved an increased level of the AMPA receptor subunit glutamate receptor 2 and synaptotagmin 1 and 2, which are pre-synaptic vesicles involved in neurotransmitter release. In addition, T3 increased dendritic spine density of principal neurons in the peri-infarct motor cortex. Moreover, we have shown that T3 regulates glutamatergic neurotransmission in cortical glutamatergic neurons. In parallel, T3 suppressed tonic GABAergic signaling in the peri-infarct tissue shown by a reduced number of parvalbumin positive neurons activity and decreased glutamic acid decarboxylase 65/67 levels. Despite TTR has been demonstrated as neuroprotective after ischemic stroke, we could not find ttr or TTR protein expression in the infarct core and peri-infarct area, and it seems unlikely that TTR is involved in mechanisms of tissue reorganization following PT during the recovery phase after stroke. Our results indicate that T3 modulates excitatory and inhibitory neurotransmission relevant for plasticity processes in the postischemic brain. T3 administration during the critical period for brain recovery regulates mechanisms that balance excitation – inhibition in favor of excitation. Further understanding and target those mechanisms might be exploited in future therapies to enhance functional recovery in stroke patients.O acidente vascular cerebral (AVC) continua a representar uma das maiores causas de morte e comorbidade a nível mundial, sendo o AVC isquémico o tipo mais comum, que representa 87% dos casos. Em Portugal o AVC é a primeira causa de morte e incapacidade em pessoas idosas. Os sintomas são variáveis de acordo com a região cerebral afetada e com e extensão do AVC, no entanto, a hemiparesia é experienciada por cerca de 75% dos doentes. A causa mais frequente de um AVC isquémico deve-se à obstrução do fluxo sanguíneo numa artéria cerebral devido à oclusão por um trombo, geralmente um ateroma ou um coágulo, ou por um êmbolo proveniente do coração ou das artérias carótidas. A extensão dos danos depende do tempo em que as células cerebrais ficam privadas de oxigénio e glicose, ou seja, em ambiente de hipóxia. No centro do enfarte, onde o fluxo sanguíneo é inexistente, há uma rápida degeneração do tecido cerebral e morte celular. A morte celular deve-se, principalmente, ao aumento do influxo de cálcio, resultante da excitotoxicidade devido a excesso de glutamato extracelular e ativação excessiva dos canais de glutamato N-metilo-D-aspartato (NMDA) e ácido alfa-amino-3-hidroxi-5-metil-4-isoxazol-propiónico (AMPA). Todavia, na região adjacente ao centro do enfarte ou na penumbra, os danos são menores e potencialmente reversíveis, devido ao fluxo sanguíneo colateral. As primeiras 4 horas e 30 minutos após o início de um AVC são cruciais para reduzir os efeitos da excitotoxicidade, edema e inflamação aguda. O tratamento com agentes fibrinolíticos ou trombectomia durante esse período após um AVC é eficaz, na medida em que restabelece o fluxo sanguíneo e previne que as células cerebrais sofram danos irreversíveis. Porém, uma minoria de doentes é elegível a este tipo de tratamento na fase aguda, o que limita a sua aplicabilidade. Atualmente, o tratamento de um AVC consiste na recanalização dos vasos obstruídos se possível, e a longo prazo na reabilitação do doente. Após um AVC, o processo de recuperação funcional ocorre com maiores progressos durante as primeiras quatro semanas e prolonga-se durante meses a anos. Durante este período, o cérebro adota diversos mecanismos de reorganização anatómica e fisiológica espontânea nas áreas subjacentes ao centro do enfarte e na região contralateral, ou seja, neuroplasticidade espontânea. No entanto, a recuperação neurológica espontânea é muito limitada, na medida em que o doente recupera apenas parcialmente algumas funções motoras. Todavia, a recuperação pode ser otimizada através de estímulos terapêuticos. Muitos esforços têm sido feitos no desenvolvimento de terapias adjuvantes que promovam a neuroplasticidade, contudo, nenhuma foi ainda aprovada em ensaios clínicos. De momento, a abordagem clínica a longo prazo em sobreviventes de um AVC continua a ser a reabilitação. As hormonas da tiroide (TH) e a sua proteína de transporte, transtirretina (TTR), são potenciais alvos terapêuticos para estimular os mecanismos endógenos de reparação neuronal. As TH desempenham funções essenciais durante o desenvolvimento cerebral e na vida adulta. Em particular a forma ativa 3,5,3’-triiodo-L-tironina (T3) é crucial na regulação de mecanismos de plasticidade neuronal, estimulação da angiogénese e neurogénese, na modulação da função de componentes do citoesqueleto e em processos de transporte intracelular. Estes mecanismos estão também presentes durante o processo de recuperação e estimulam a recuperação da função motora durante as primeiras semanas a meses após um AVC. Independentemente da sua função como proteína de transporte, a TTR tem sido estudada como uma molécula neuroprotetora no cérebro e posta em evidência como alvo promissor durante a fase de recuperação após um AVC, de forma a melhorar as funções neurológicas perdidas. Os ensaios experimentais apresentados nesta tese tiveram como objetivo investigar o papel das TH e da TTR na reorganização da função neuronal após fototrombose (PT). A administração de T3 a 50 μg/kg durante as primeiras duas semanas após a indução experimental de AVC, realizada por PT em murganhos, melhorou significativamente a recuperação da função neurológica perdida sem afetar o volume do enfarte. Observámos uma recuperação da função motora, acompanhada por mecanismos de regulação homeostática na periferia do enfarte, em favor da excitabilidade. A nível celular e estrutural demonstramos que a T3 tem efeitos modulatórios que atuam a diferentes escalas temporais e locais, de forma a assegurar uma eficiente neurotransmissão sináptica. Mostrámos também que a administração a longo prazo da T3, após PT, induz alterações estruturais ao aumentar a densidade das espículas dendríticas na periferia do enfarte e na região contralateral. A eficácia da neurotransmissão sináptica parece ter sido aumentada, após administração da T3, devido ao aumento dos níveis de sinaptotagmina 1 e 2, que são proteínas vesiculares pré-sinápticas envolvidas na libertação de neurotransmissores e ao aumento dos níveis da subunidade 2 dos recetores de glutamato AMPA, na periferia do enfarte. A administração da T3 também diminui a ação inibitória do neurotransmissor ácido gama-aminobutírico (GABA) na periferia do enfarte devido à redução da atividade dos neurónios que expressam a parvalbumina e à redução dos níveis da descarboxilase do glutamato (GAD) 65/67. Além disso, mostrámos que a T3 modula, in vitro, propriedades intrínsecas da membrana neuronal, com o equilíbrio das correntes dos recetores ionotrópicos ativados pelo glutamato e diminuição dos níveis de sinaptotagmina em neurónios submetidos a privação de oxigénio e glicose. Curiosamente, encontrámos níveis aumentados do recetor TRβ1, que medeia as ações da T3, no centro do enfarte de amostras post-mortem de doentes que sofreram um AVC. Apesar do seu já demonstrado efeito neuroprotetor, não encontramos expressão do gene nem da proteína TTR no córtex cerebral de murganhos nas duas semanas após PT e, temos dúvidas que a TTR participe em mecanismos de recuperação motora após um AVC. Em conclusão, os nossos resultados indicam que a T3 modula a neurotransmissão, excitatória e inibitória, relevante para os processos de plasticidade neuronal após um AVC. A administração da T3 durante o período crítico para a recuperação regula mecanismos que equilibram o rácio excitação – inibição, em favor da excitação. Nesse contexto, os resultados apresentados parecem muito promissores, no sentido de serem explorados em futuros ensaios clínicos, de forma a desenvolver novas terapias para melhorar a recuperação motora em doentes que sofreram um AVC

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 TRβ1 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.info:eu-repo/semantics/publishedVersio

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

Contribution to the study of feline pemphigus foliaceus

Dissertação de Mestrado Integrado em Medicina Veterinária, Ciências VeterináriasO pênfigo foliáceo é a doença de pele autoimune mais comum no gato, não estando descrita predisposição etária, genética ou sexual. Neste trabalho, foi realizado um estudo retrospectivo de onze casos diagnosticados de pênfigo foliáceo na espécie felina, durante um período de três anos na clínica ADVETIA. O início das lesões variou de menos de 1 ano a 10 anos de idade, mediana de 5 anos. Os sinais clínicos observados incluíram crostas (n=11), paroníquia (n=7), erosões (n=4) e alopécia (n=4). As lesões foram mais frequentes nas pregas ungueais (n=7), pavilhão auricular externo (n=7) e/ou pavilhão auricular interno (n=3), plano nasal (n=3) e nariz (n=3). A avaliação citológica em 7 casos demonstrou a presença de queratinócitos acantolíticos. No exame histológico de 4 biópsias observaram-se queratinócitos acantolíticos e crostas neutrofílicas (n=2). Num caso não foi observado acantólise activa. Todos os casos foram seguidos por 1 a 49 meses. A maioria dos casos (n=9) atingiram remissão completa com tratamento imunosupressor. Apenas um gato foi eutanasiado, devido ao desenvolvimento de sinais sistémicos durante a terapia.Pemphigus foliaceus is the most common auto-immune skin disease in cats. No age, breed or sex predisposition are reported. A retrospective study of eleven cats diagnosed for pemphigus foliaceus over a period of three years in “ADVETIA” clinic was performed. Age at onset ranged from less than 1 year to 10 years, median 5 years. Clinical signs of the disease included crusts (n=11), paronychia (n=7), erosions (n=4) and alopecia (n=4). Lesions were most common on claw folds (n=7), outer pinnae (n=7) and/or inner pinnae (n=3), nasal planum (n=3) and muzzle (n=3). Cytologic evaluation revealed acantholytic keratinocytes in 7 cats. The histological examination of 4 biopsy specimens revealed acantholytic keratinocytes and neutrophilic crusts (n=2). Active acantholysis was not observed in 1 case. All cases were followed for 1 to 49 months. Most cats (n=9) achieved total remission with immunosuppressive treatment. Only one cat died due to systemic signs development during therapy

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

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

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 ar-tery (tMCAO) for 120 min. This response was also found in rats subjected to permanent focal ische-mia 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 with-out treatment effect. The results provide the first evidence of the plasticity-promoting actions of dopaminergic treatment following stroke

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

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