Astrocytes posses a wide range of functions within the brain. In response to ischemic conditions they swell due to increased uptake of osmolytes and they are mainly responsible for cytotoxic edema formation. However, they are also able to regulate their volume by releasing osmolytes together with water via the process of regulatory volume decrease (RVD). The Aquaporin 4 (AQP4) channel and Transient receptor potential vanilloid 4 (TRPV4) channel are suspected to be strongly involved in these processes of astrocytic volume regulation. The goal of the present diploma thesis was to clarify the role of both channels in astrocytic swelling in situ. For our experiments we used a subpopulation of green fluorescent protein-labelled astrocytes from AQP4-deficient (AQP4-/- ), TRPV4-deficient (TRPV4-/- ) and control (Ctrl) mice. Cell volume alterations were induced in acute brain slices by hypoosmotic stress or by oxygen-glucose deprivation (OGD). Data were quantified using fluorescence intensity-based approach in the whole cells and in astrocytic endfeet. Our results indicate, that there is no difference in astrocytic swelling or cell volume recovery between astrocytes from AQP4-/- , TRPV4-/- and control mice when exposed to hypoosmotic stress. On the contrary, volume changes induced by OGD varied...

Spinal cord stimulation (SCS) inhibits substance P (SP) release and decreases the expression of the transient receptor potential vanilloid 1 (TRPV1) in the spinal cord at thoracic 4 (T4) during cardiac ischemia in rat models (. Ding et al., 2007). We hypothesized that activation of TRPV1 in the T4 spinal cord segment by intermittent occlusion of the left anterior descending coronary artery (CoAO) mediates spinal cord SP release. Experiments were conducted in urethane-anesthetized Sprague-Dawley male rats using SP antibody-coated microprobes to measure SP release at the central terminal endings of cardiac ischemic-sensitive afferent neurons (CISAN) in the spinal T4 dorsal horns. Vehicle, capsaicin (CAP; TRPV1 agonist) and capsazepine (CZP; TRPV1 antagonist) were injected into the left T4 prior to stimulation of CISAN by intermittent CoAO (with or without upper cervical SCS). CAP induced endogenous SP release from laminae I and II in the T4 spinal cord above baseline. Conversely, CZP injections significantly inhibited SP release from laminae I-VII in the T4 spinal cord segment below baseline. CZP also attenuated CoAO-induced SP release, while T4 injections of CZP with SCS completely restored SP release to basal levels during CoAO activation. CAP increased the number of c-Fos (a marker for CISAN activation) positive T4 dorsal horn neurons compared to sham-operated animals, while CZP (alone or during CoAO and SCS. +. CoAO) significantly reduced the number of c-Fos positive neurons. These results suggest that spinal release of the putative nociceptive transmitter SP occurs, at least in part, via a TRPV1 mechanism.

Introdução: Receptores TRPV1 são canais catiônicos nociceptivos não seletivos. Eles desempenham um papel na sensação de dor na córnea. No presente trabalho, descreveremos a resposta do TRPV1 na córnea de camundongos e TRPV1 -/- a estímulos químicos e o efeito do SNAP sobre este receptor. Métodos: camundongos C57BL/6 e TRPV1 -/- foram comparados. As observações incluíram o influxo de cálcio na cultura, observação direta e o comportamento de limpeza do olho após o desafio na córnea com 1µM da capsaicina (CAP) com ou sem 1mM do colírio SNAP. Resultados: As características da superfície ocular não eram diferentes entre ambos os genótipos, exceto que a sensibilidade para CAP é inferior em TRPV1 -/- (p = 0,0019 e 0,0095, respectivamente). A imunocoloração revelou que TRPV1 é expresso na camada basal do epitélio da córnea, apenas nos camundongos C57BL/6. CAP estimula o influxo de cálcio em cultura de células epiteliais e a sensibilidade corneana em C57BL/6 in vivo foi reduzida na presença do SNAP (p = 0,0329 e 0,01, respectivamente). Conclusão: A ausência dos receptores TRPV1 no epitélio da córnea não afeta o fenótipo da superfície ocular de camundongos. A resposta para tirar colírios revelou que ela poderia funcionar como uma terapia analgésica devido ao seu antagonismo com o TRPV1. / TRPV-1 receptors are non-selective nocioceptive cation channels. They play a role in cornea sensation. In the present work we describe the response of corneas of TRPV1-/- and mice to chemical stimuli and the effect of SNAP on this receptor. TRPV1-/- and C57BL/6 mice were compared. The observations included the calcium influx in culture, direct observation and the behavior of eye wipe after corneal challenge with 1?M Capsaicin (CAP) with or not 1mM SNAP eye drops. Ocular surface characteristics were not different between both genotypes, except that the sensitivity to CAP is lower in TRPV1-/- (p=0.0019 and 0.0095, respectively). Immunostaining revealed that TRPV 1 is expressed in the basal layer of the cornea epithelia, only in the C57 mice. CAP stimulated calcium influx in epithelial cells in culture and cornea sensitivity in C57 mice in vivo was reduced in the presence of SNAP (p=0.0329 and 0.01, respectively). The absence of TRPV-1 receptors in the cornea epithelia did not affect the ocular surface phenotype in mice. The response to SNAP eye drops revealed that it could work as an analgesic therapy due to its antagonism to the TRPV1.

Introdução: Receptores TRPV1 são canais catiônicos nociceptivos não seletivos. Eles desempenham um papel na sensação de dor na córnea. No presente trabalho, descreveremos a resposta do TRPV1 na córnea de camundongos e TRPV1 -/- a estímulos químicos e o efeito do SNAP sobre este receptor. Métodos: camundongos C57BL/6 e TRPV1 -/- foram comparados. As observações incluíram o influxo de cálcio na cultura, observação direta e o comportamento de limpeza do olho após o desafio na córnea com 1µM da capsaicina (CAP) com ou sem 1mM do colírio SNAP. Resultados: As características da superfície ocular não eram diferentes entre ambos os genótipos, exceto que a sensibilidade para CAP é inferior em TRPV1 -/- (p = 0,0019 e 0,0095, respectivamente). A imunocoloração revelou que TRPV1 é expresso na camada basal do epitélio da córnea, apenas nos camundongos C57BL/6. CAP estimula o influxo de cálcio em cultura de células epiteliais e a sensibilidade corneana em C57BL/6 in vivo foi reduzida na presença do SNAP (p = 0,0329 e 0,01, respectivamente). Conclusão: A ausência dos receptores TRPV1 no epitélio da córnea não afeta o fenótipo da superfície ocular de camundongos. A resposta para tirar colírios revelou que ela poderia funcionar como uma terapia analgésica devido ao seu antagonismo com o TRPV1. / TRPV-1 receptors are non-selective nocioceptive cation channels. They play a role in cornea sensation. In the present work we describe the response of corneas of TRPV1-/- and mice to chemical stimuli and the effect of SNAP on this receptor. TRPV1-/- and C57BL/6 mice were compared. The observations included the calcium influx in culture, direct observation and the behavior of eye wipe after corneal challenge with 1?M Capsaicin (CAP) with or not 1mM SNAP eye drops. Ocular surface characteristics were not different between both genotypes, except that the sensitivity to CAP is lower in TRPV1-/- (p=0.0019 and 0.0095, respectively). Immunostaining revealed that TRPV 1 is expressed in the basal layer of the cornea epithelia, only in the C57 mice. CAP stimulated calcium influx in epithelial cells in culture and cornea sensitivity in C57 mice in vivo was reduced in the presence of SNAP (p=0.0329 and 0.01, respectively). The absence of TRPV-1 receptors in the cornea epithelia did not affect the ocular surface phenotype in mice. The response to SNAP eye drops revealed that it could work as an analgesic therapy due to its antagonism to the TRPV1.

Pain is the most feared symptom of cancer and can impact patients' lives more than the cancer itself. Despite improvements in cancer prevention and detection, pain is often the first sign of cancer, with an estimated 70-75% of advanced stage cancer patients presenting with skeletal metastases. Cancer metastasis to the bone is associated with persistent pain that increases in intensity over time. Current treatments follow the World Health Organization (WHO) analgesic ladder for cancer pain management suggesting non-steroidal anti-inflammatory drugs (NSAIDs) for mild to moderate pain and opioids for moderate to severe pain. However, estimates indicate as many as 50-80% of cancer patients worldwide receive inadequate pain management. Moreover, opioid doses required for these patients are associated with adverse side effects further diminishing quality of life. Development of improved non-opioid therapies is dependent on increased understanding of mechanisms driving cancer pain and its relief. The objective of this dissertation was to characterize a rat model of cancer-induced bone pain, to develop approaches to measure both ongoing and breakthrough pain and to investigate the contribution of underlying inflammatory mechanisms to pain, bone destruction and bone remodeling. Using female Fischer F344/NhSD rats, histocompatible MAT B III mammary adenocarcinoma cells were sealed into the intramedullary space of the right rear tibia for a time course of 13 days. Ongoing pain was characterized based on the WHO 3-step ladder for pain management utilizing novel behavioral and neurochemical assays. Morphine and peripheral nerve block were sufficient to control ongoing pain, whereas NSAID treatment failed to provide pain relief. Cancer-bearing rats selectively displayed movement-induced breakthrough pain to a background of morphine-controlled ongoing pain. Furthermore, we determined that breakthrough pain is initiated, but not maintained, by peripheral afferent input from the tumor-bearing tibia using lidocaine administration prior to or following movement. For the final part of this study, we investigated the role of transient receptor potential vanilloid 1 (TRPV1) and interleukin-6 (IL-6) blockade, as these have been shown to be important mediators in animal models CIBP. Acute blockade of TRPV1 channels by AMG9810 selectively reversed inflammatory-induced pain, but failed to control evoked or ongoing CIBP. Acute blockade of interleukin-6 signaling by TB-2-081, an IL-6 receptor antagonist, successfully reversed evoke pain responses, but like AMG9810, failed to control ongoing pain. Sustained administration of TB-2-081 reversed cancer-induced tactile hypersensitivity and tumor-induced bone remodeling of the tibia. Further in vitro analysis revealed TB-2-081 functions by inhibiting the Jak/STAT cascade on both tumor cells and osteoblasts, suggesting that blockade of IL-6 signaling can effectively modulate the bone microenvironment to reduce tumor burden and pain. Combined, our data introduce a rat model of breast cancer bone metastasis, in which the underlying mechanisms of ongoing and breakthrough CIBP can be effectively studied. From this, novel therapeutic agents can be developed and investigated to help improve quality of life in patients suffering from this disease.

TRPV channels play a role in both mammalian insulin signaling, with TRPV1 expression in pancreatic beta-cells, and in C. elegans insulin-like signaling through expression of OSM-9, OCR-1, and OCR-2 in stress response pathways. In response to a glucose-supplemented diet, C. elegans are know to have sensitivity to anoxic stress, exhibit chemotaxis attraction, and display reduced egg-laying rate. Transcriptome analysis reveals that glucose stimulates nervous system activity with increased transcript levels of genes regulating neurotransmitters. Ciliated sensory neurons are needed for a reduced egg-laying phenotype on a glucose-supplemented diet. Egg-laying rate is not affected when worms graze on glucose-supplemented Delta-PTS OP50 E. coli, which is defective in glucose uptake. This suggests a possible sensory neuron obstruction by exopolysaccharides produced by standard OP50 E. coli on glucose, eliciting a starvation response from the worm and causing reduced egg-laying rate. Glucose chemotaxis is affected in specific TRPV subunit allele mutants: ocr-2(vs29) and osm-9(yz6), serotonin receptor mutants: ser-1(ok345) and mod-1(ok103), and G-alpha protein mutant: gpa-10(pk362). TRPV deletion mutants had no effect on glucose chemotaxis, alluding to the modality role pf TRPV alleles in specific sensory neurons. The role of serotonin in a reduced egg-laying rate with glucose remains unclear.

Spinal cord stimulation (SCS) is used to reduce angina that accompanies cardiac ischemia, but little is known about the molecular mechanisms mediating this effect. We studied the expression of SP, neurokinin-1 (NK-1) receptor, and transient receptor potential vanilloid type 1 (TRPV1) mRNA in the rat spinal cord at thoracic 4 (T4), cervical 2 (C2) and caudal brain stem by RT-PCR during intermittent occlusion of the left anterior descending coronary artery (CoAO), during sustained SCS by itself at the C2 spinal segment, and during sustained SCS plus intermittent CoAO. Only SP mRNA was increased significantly in T4 and brainstem during CoAO, while SCS decreased the mRNA levels of SP, NK-1 and TRPV1 significantly in T4 and the brainstem. SCS attenuated the increase of SP and TRPV1 mRNA levels at T4 level induced by intermittent CoAO when the stimulation was applied prior to the initiation of the cardiac ischemia. These results support the role for SP as a putative neurotransmitter for the myocardial ischemia-sensitive afferent neuron signal to the spinal level. They suggest that modification of the ischemic cardiac nociceptive afferent signal by SCS involves a change in SP and TRPV1 expression.

<p>The rotator cuff is a group of four muscles and tendons in the shoulder that function to lift and rotate the arm. Rotator cuff tendon tears are increasingly common: more than 545,000 rotator cuff surgeries occur annually in the US. However, treatment is often complicated by disorganized collagen matrix formed via fibrosis and results in high re-tear rates. Tendon tissue engineering seeks to solve the problem using biomaterials to promote neo-tendon formation to augment repair or regenerate tendon. However, while current biomaterials provide the opportunity to improve tendon healing, they frequently still exhibit fibrosis in preclinical studies. Therefore, a critical need exists to understand the mechanisms of aligned collagen formation when designing biomaterials for tendon tissue engineering. Matrix architecture and transient receptor potential cation channel subfamily V member 4 (TRPV4) regulate aligned collagen formation during tenogenesis in vitro, but the mechanism remains to be determined. Recently, TRPV4 stimulation was found to induce nuclear localization and activation of transcriptional co-activators Yes-associated protein (YAP). YAP expression is upregulated during tendon development, a process characterized by aligned collagen formation, and in response to physiological mechanical stimulation, suggesting it could play an important role in tendon. The objective of this work is to improve tissue engineering strategies and progress toward making a device that regenerate tendon after injury. Aim 1 incorporates tendon-derived matrix into synthetic polymer scaffolds to add biological signaling cues to induce tenogenesis. Aim 2 uses a 2D photolithography system (microphotopatterning) to optimize architectural and structural cues to promote stem cell differentiation toward tenogenic, chondrogenic, and osteogenic lineages. Aim 3 investigates dynamic tensile loading protocols to promote collagen matrix synthesis and improve engineered tendon mechanical function. Aim 4 investigates the role of TRPV4 and YAP in collagen alignment during engineered tendon development.</p>