Modulation of mechanosensory vibrissal responses in the trigeminocervical complex by stimulation of the greater occipital nerve in a rat model of trigeminal neuropathic pain.

Background Stimulation of the occipital or trigeminal nerves has been successfully used to treat chronic refractory neurovascular headaches such as migraine or cluster headache, and painful neuropathies. Convergence of trigeminal and occipital sensory afferents in the ‘trigeminocervical complex’ (TCC) from cutaneous, muscular, dural, and visceral sources is a key mechanism for the input-induced central sensitization that may underlie the altered nociception. Both excitatory (glutamatergic) and inhibitory (GABAergic and glycinergic) mechanisms are involved in modulating nociception in the spinal and medullary dorsal horn neurons, but the mechanisms by which nerve stimulation effects occur are unclear. This study was aimed at investigating the acute effects of electrical stimulation of the greater occipital nerve (GON) on the responses of neurons in the TCC to the mechanical stimulation of the vibrissal pad. Methods Adult male Wistar rats were used. Neuronal recordings were obtained in laminae II-IV in the TCC in control, sham and infraorbital chronic constriction injury (CCI-IoN) animals. The GON was isolated and electrically stimulated. Responses to the stimulation of vibrissae by brief air pulses were analyzed before and after GON stimulation. In order to understand the role of the neurotransmitters involved, specific receptor blockers of NMDA (AP-5), GABAA (bicuculline, Bic) and Glycine (strychnine, Str) were applied locally. Results GON stimulation produced a facilitation of the response to light facial mechanical stimuli in controls, and an inhibition in CCI-IoN cases. AP-5 reduced responses to GON and vibrissal stimulation and blocked the facilitation of GON on vibrissal responses found in controls. The application of Bic or Str significantly reduced the facilitatory effect of GON stimulation on the response to vibrissal stimulation in controls. However, the opposite effect was found when GABAergic or Glycinergic transmission was prevented in CCI-IoN cases. Conclusions GON stimulation modulates the responses of TCC neurons to light mechanical input from the face in opposite directions in controls and under CCI-IoN. This modulation is mediated by GABAergic and Glycinergic mechanisms. These results will help to elucidate the neural mechanisms underlying the effectiveness of nerve stimulation in controlling painful craniofacial disorders, and may be instrumental in identifying new therapeutic targets for their prevention and treatment.post-print1932 K

Microglia and Inhibitory Circuitry in the Medullary Dorsal Horn: Laminar and Time-Dependent Changes in a Trigeminal Model of Neuropathic Pain.

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Chronic administration of P2X7 receptor antagonist JNJ-47965567 delays disease onset and progression, and improves motor performance in ALS SOD1 (G93A) female mice

Neuroinflammation is one of the main physiopathological mechanisms of amyotrophic lateral sclerosis (ALS), produced by the chronic activation of microglia in the CNS. This process is triggered by the persistent activation of the ATP-gated P2X7 receptor (P2RX7, hereafter referred to as P2X7R). The present study aimed to evaluate the effects of the chronic treatment with the P2X7R antagonist JNJ-47965567 in the development and progression of ALS in the SOD1 murine model. SOD1 mice were intraperitoneally (i.p.) injected with either 30 mg/kg of JNJ-47965567 or vehicle 4 times per week, from pre-onset age (here, postnatal day 60; P60) until study endpoint. Body weight, motor coordination, phenotypic score, disease onset and survival were measured throughout the study, and compared between vehicle- and drug-injected groups. Treatment with the P2X7R antagonist JNJ-47965567 delayed disease onset, reduced body weight loss and improved motor coordination and phenotypic score in female SOD1 mice, although it did not increase lifespan. Interestingly, neither beneficial nor detrimental effects were observed in males in any of the analyzed parameters. Treatment did not affect motor neuron survival or ChAT, Iba-1 and P2X7R protein expression in endpoint individuals of mixed sexes. Overall, chronic administration of JNJ-47965567 for 4 times per week to SOD1 mice from pre-onset stage altered disease progression in female individuals while it did not have any effect in males. Our results suggest a partial, yet important, effect of P2X7R in the development and progression of ALS. G93A G93A G93A G93AThis project was supported by the European Union’s Horizon 2020 research and innovation programme under Marie Sklodowska-Curie grant agreement no. 766124; the Spanish Ministry of Science, Technology and Innovation (SAF2016-78892R); and Fundación Teófilo Hernando

Multiple Morphometric Assessment of Microglial Cells in Deafferented Spinal Trigeminal Nucleus.

Microglia (MG) are the first cells to react to the abnormal incoming signals that follow an injury of sensory nerves and play a critical role in the development and maintenance of neuropathic pain, a common sequel of nerve injuries. Here we present population data on cell number, soma size, and length of processes of MG in the caudal division of the spinal trigeminal nucleus (Sp5C) in control mice and at the peak of microgliosis (7 days) following unilateral transection of the infraorbital nerve (IoN). The study is performed combining several bias- and assumption-free imaging and stereological approaches with different immunolabeling procedures, with the objective of tackling some hard problems that often hinder proper execution of MG morphometric studies. Our approach may easily be applied to low-density MG populations, but also works, with limited biases, in territories where MG cell bodies and processes form dense meshworks. In controls, and contralaterally to the deafferented side, MG cell body size and shape and branching pattern matched well the descriptions of “resting” or “surveillant” MG described elsewhere, with only moderate intersubject variability. On the superficial laminae of the deafferented side, however, MG displayed on average larger somata and remarkable diversity in shape. The number of cells and the length of MG processes per mm3 increased 5 and 2.5 times, respectively, indicating a net 50% decrease in the mean length of processes per cell. By using specific immunolabeling and cell sorting of vascular macrophages, we found only a negligible fraction of these cells in Sp5C, with no differences between controls and deafferented animals, suggesting that blood-borne monocytes play at most a very limited role in the microgliosis occurring following sensory nerve deafferentation. In sum, here we present reliable morphometric data on MG in control and deafferented trigeminal nuclei using efficient methods that we propose may equally be applied to any morphometric population analysis of these cells under different physiological or pathological conditions.post-print1841 K

Response Facilitation Induced by Insulin-like Growth Factor-I in the Primary Somatosensory Cortex of MiceWas Reduced in Aging.

Aging is accompanied by a decline in cognition that can be due to a lower IGF-I level. We studied response facilitation induced in primary somatosensory (S1) cortical neurons by repetitive stimulation of whiskers in young and old mice. Layer 2/3 and 5/6 neurons were extracellularly recorded in young ( 6 months of age) and old ( 20 month of age) anesthetized mice. IGF-I injection in S1 cortex (10 nM; 0.2 L) increased whisker responses in young and old animals. A stimulation train at 8 Hz induced a long-lasting response facilitation in only layer 2/3 neurons of young animals. However, all cortical neurons from young and old animals showed long-lasting response facilitation when IGF-I was applied in the S1 cortex. The reduction in response facilitation in old animals can be due to a reduction in the IGF-I receptors as was indicated by the immunohistochemistry study. Furthermore, a reduction in the performance of a whisker discrimination task was observed in old animals. In conclusion, our findings indicate that there is a reduction in the synaptic plasticity of S1 neurons during aging that can be recovered by IGF-I. Therefore, it opens the possibility of use IGF-I as a therapeutic tool to ameliorate the effects of heathy aging.post-print2685 K

Implication of type 4 NADPH oxidase (NOX4) in tauopathy.

Aggregates of the microtubule-associated protein tau are a common marker of neurodegenerative diseases collectively termed as tauopathies, such as Alzheimer’s disease (AD) and frontotemporal dementia. Therapeutic strategies based on tau have failed in late stage clinical trials, suggesting that tauopathy may be the consequence of upstream causal mechanisms. As increasing levels of reactive oxygen species (ROS) may trigger protein aggregation or modulate protein degradation and, we had previously shown that the ROS producing enzyme NADPH oxidase 4 (NOX4) is a major contributor to cellular autotoxicity, this study was designed to evaluate if NOX4 is implicated in tauopathy. Our results show that NOX4 is upregulated in patients with frontotemporal lobar degeneration and AD patients and, in a humanized mouse model of tauopathy induced by AVV-TauP301L brain delivery. Both, global knockout and neuronal knockdown of the Nox4 gene in mice, diminished the accumulation of pathological tau and positively modified established tauopathy by a mechanism that implicates modulation of the autophagy-lysosomal pathway (ALP) and, consequently, improving the macroautophagy flux. Moreover, neuronal-targeted NOX4 knockdown was sufficient to reduce neurotoxicity and prevent cognitive decline, even after induction of tauopathy, suggesting a direct and causal role for neuronal NOX4 in tauopathy. Thus, NOX4 is a previously unrecognized causative, mechanism-based target in tauopathies and blood-brain barrier permeable specific NOX4 inhibitors could have therapeutic potential even in established disease.post-print5895 K

Response Facilitation Induced by Insulin-like Growth Factor-I in the Primary Somatosensory Cortex of Mice Was Reduced in Aging

Aging is accompanied by a decline in cognition that can be due to a lower IGF-I level. We studied response facilitation induced in primary somatosensory (S1) cortical neurons by repetitive stimulation of whiskers in young and old mice. Layer 2/3 and 5/6 neurons were extracellularly recorded in young (≤ 6 months of age) and old (≥ 20 month of age) anesthetized mice. IGF-I injection in S1 cortex (10 nM; 0.2 μL) increased whisker responses in young and old animals. A stimulation train at 8 Hz induced a long-lasting response facilitation in only layer 2/3 neurons of young animals. However, all cortical neurons from young and old animals showed long-lasting response facilitation when IGF-I was applied in the S1 cortex. The reduction in response facilitation in old animals can be due to a reduction in the IGF-I receptors as was indicated by the immunohistochemistry study. Furthermore, a reduction in the performance of a whisker discrimination task was observed in old animals. In conclusion, our findings indicate that there is a reduction in the synaptic plasticity of S1 neurons during aging that can be recovered by IGF-I. Therefore, it opens the possibility of use IGF-I as a therapeutic tool to ameliorate the effects of heathy aging

El Complejo Trigeminocervical del roedor. Enclave neural del dolor neuropático craneofacial

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Anatomía, Histología y Neurociencia. Fecha de lectura: 22-02-2021Esta tesis tiene embargado el acceso al texto completo hasta el 22-08-2022Los síndromes dolorosos craneofaciales incluyen una serie de afecciones que constituyen un importante problema de salud pública, sin embargo, su fisiopatología es, en gran medida, desconocida. En esta Tesis Doctoral hemos abordado el estudio de diversos sustratos neurales claves en el mantenimiento del dolor neuropático trigeminal en el Complejo Trigeminocervical (TCC), área de convergencia entre los aferentes trigeminales y cervicales, en un modelo de dolor neuropático ampliamente utilizado en roedores como es la constricción crónica del nervio infraorbitario (CCI-IoN). Para ello hemos utilizado técnicas de inmunomarcado, microscopía electrónica, electrofisiología y cuantificación estereológica. En este trabajo, hemos comenzado realizando una descripción completa y actual de la anatomía del nervio occipital mayor (NOM) y sus conexiones centrales. Centrándonos a continuación, en tres elementos claves en la generación y mantenimiento del dolor craneocefálico como son las aferentes primarias, la microglia (MG) y las interneuronas inhibitorias locales. Los resultados muestran que el NOM es un nervio sensitivo en su recorrido superficial en la región occipital, formando por entre 1 y 4 fascículos que contienen un promedio de 900 axones mielínicos y 3300 amielínicos de los cuales más del 90% emergen de las neuronas sensoriales primarias que se encuentran en C2 y el 10% restante emerge de C3. Las proyecciones centrales del NOM siguen una distribución laminar y morfológica compleja, convergiendo algunas de ellas con los aferentes provenientes del ganglio trigémino en el TCC. Los resultados electrofisiológicos revelan que la estimulación del NOM modula, mediante mecanismos GABAérgicos y Glicinérgicos, las respuestas neuronales a la estimulación táctil inocua de las vibrisas facilitándola en animales control e inhibiéndola en animales CCI-IoN. La MG, que constituye un elemento dinámico y central en el desarrollo del dolor neuropático, mostró un proceso de activación inducido por CCI-IoN, con un pico de microgliosis 5 días después de la lesión que a los 21 días se había reducido considerablemente, aunque siguió siendo significativa comparada con los animales control. Además, el estudio, combinando varios enfoques estereológicos, nos permitió obtener datos morfométricos fiables de las células MG de forma insesgada. Estos hallazgos ayudarán a comprender mejor las bases neurales que, en los territorios de convergencia sensorial en los sistemas espinomedulares, subyacen a la derivación y diseminación del dolor en los trastornos craneofaciales dolorosos grave

Implication of type 4 NADPH oxidase (NOX4) in tauopathy

Aggregates of the microtubule-associated protein tau are a common marker of neurodegenerative diseases collectively termed as tauopathies, such as Alzheimer's disease (AD) and frontotemporal dementia. Therapeutic strategies based on tau have failed in late stage clinical trials, suggesting that tauopathy may be the consequence of upstream causal mechanisms. As increasing levels of reactive oxygen species (ROS) may trigger protein aggregation or modulate protein degradation and, we had previously shown that the ROS producing enzyme NADPH oxidase 4 (NOX4) is a major contributor to cellular autotoxicity, this study was designed to evaluate if NOX4 is implicated in tauopathy. Our results show that NOX4 is upregulated in patients with frontotemporal lobar degeneration and AD patients and, in a humanized mouse model of tauopathy induced by AVV-TauP301L brain delivery. Both, global knockout and neuronal knockdown of the Nox4 gene in mice, diminished the accumulation of pathological tau and positively modified established tauopathy by a mechanism that implicates modulation of the autophagy-lysosomal pathway (ALP) and, consequently, improving the macroautophagy flux. Moreover, neuronal-targeted NOX4 knockdown was sufficient to reduce neurotoxicity and prevent cognitive decline, even after induction of tauopathy, suggesting a direct and causal role for neuronal NOX4 in tauopathy. Thus, NOX4 is a previously unrecognized causative, mechanism-based target in tauopathies and blood-brain barrier permeable specific NOX4 inhibitors could have therapeutic potential even in established disease