Brazilian National Network of Alternative Methods (RENAMA) 10th Anniversary: Meeting of the Associated Laboratories, May 2022.

The Brazilian National Network of Alternative Methods (RENAMA), which is linked to the Ministry of Science, Technology and Innovation, is currently comprised of 51 laboratories from CROs, academia, industry and government. RENAMA's aim is to develop and validate new approach methodologies (NAMs), as well as train researchers and disseminate information on their use - thus reducing Brazilian, and consequently Latin American, dependence on external technology. Moreover, it promotes the adoption of NAMs by educators and trained researchers, as well as the implementation of good laboratory practice (GLP) and the use of certified products. The RENAMA network started its activities in 2012, and was originally comprised of three central laboratories - the National Institute of Metrology, Quality and Technology (INMETRO); the National Institute of Quality Control in Health (INCQS); and the National Brazilian Biosciences Laboratory (LNBio) - and ten associated laboratories. In 2022, RENAMA celebrated its 10th anniversary, a milestone commemorated by the organisation of a meeting attended by different stakeholders, including the RENAMA-associated laboratories, academia, non-governmental organisations and industry. Ninety-six participants attended the meeting, held on 26 May 2022 in Balneário Camboriú, SC, Brazil, as part of the programme of the XXIII Brazilian Congress of Toxicology 2022. Significant moments of the RENAMA were remembered, and new goals and discussion themes were established. The lectures highlighted recent innovations in the toxicological sciences that have translated into the assessment of consumer product safety through the use of human-relevant NAMs instead of the use of existing animal-based approaches. The challenges and opportunities in accepting such practices for regulatory purposes were also presented and discussed.

CALU-3 lung cells three-dimensionally assembled onto CellFate® matrix present angiotensin-converting enzyme-2 activity.

Currently, there is a great need for the development of three-dimensional (3D) in vitro lung models. Particularly, the production of a suitable 3D model of pulmonary epithelium for understanding the pathophysiology of diseases such as the COVID-19 must consider the tissue architecture and presence, for example, of the angiotensin-converting enzyme-2 (ACE-2) in the cells. Different polymeric membranes are being used to support cell culturing, especially of lung cells, however, there is still no information about the culture of these cells onto bacterial nanocellulose (BNC) matrices. We have used the BNC matrix CellFate® as a support for the assembly of a 3D in vitro model of lung epithelium, composed of human lung fibroblasts (HLF) and lung adenocarcinoma cells (CALU-3). CellFate® matrices were made from bacterial fermentation resulting in a natural and biocompatible biopolymer. Cells were cultured onto CellFate® and maintained in a 5% CO2 humidified atmosphere at 37°C. Cell viability was assessed by the resazurin method The samples were, then, exposed to the air-liquid interface (ALI), and histologically analyzed. ACE-2 activity was verified on the hydrolyze of the fluorogenic substrate Mca-APK(Dnp)-OH, and its presence was evaluated by flow cytometry. The expression of the anionic transporter SLCO3A1 was evaluated by qPCR. Cell viability analysis indicates that CellFate® was not toxic to these cells. By flow cytometry, the presence of the ACE-2 was identified in the CALU-3 cells surface corroborating the results obtained from enzymatic activity analysis. The SLCO3A1 transporter expression was identified in cells cultured onto CellFate®, but not in cells cultured onto the transwell (control). CALU-3 cells cultivated onto CellFate® resulted in a pseudostratified organization, a typical morphology of the human respiratory tract epithelium. The current model opens perspectives for studies involving physiological characterization, improving its relevance for the understanding of the pathophysiology of diseases as well as the response to drugs.

Cell viability and cytotoxicity of inkjet-printed flexible organic electrodes on parylene C.

This study reports on the fabrication of biocompatible organic devices by means of inkjet printing with a novel combination of materials. The devices were fabricated on Parylene C (PaC), a biocompatible and flexible polymer substrate. The contact tracks were inkjet-printed using a silver nanoparticle ink, while the active sites were inkjet-printed using a poly (3,4ethylenedioxythiophene)/polystyrene sulfonate (PEDOT:PSS) solution. To insulate the final device, a polyimide ink was used to print a thick film, leaving small open windows upon the active sites. Electrical characterization of the final device revealed conductivities in the order of 103 and 102 S.cm-1 for Ag and PEDOT based inks, respectively. Cell adhesion assays performed with PC-12 cells after 96 h of culture, and B16F10 cells after 24 h of culture, demonstrated that the cells adhered on top of the inks and cell differentiation occurred, which indicates Polyimide and PEDOT:PSS inks are non-toxic to these cells. The results indicate that PaC, along with its surface-treated variants, is a potentially useful material for fabricating cell-based microdevices.

Perspective from developers: Tissue-engineered products for skin wound healing.

Tissue-engineered products (TEPs) are at the forefront of developmental medicines, precisely where monoclonal antibodies and recombinant cytokines were 30 years ago. TEPs development for treating skin wounds has become a fast-growing field as it offers the potential to find novel therapeutic approaches for treating pathologies that currently have limited or no effective alternatives. This review aims to provide the reader with the process of translating an idea from the laboratory bench to clinical practice, specifically in the context of TEPs designing for skin wound healing. It encompasses historical perspectives, approved therapies, and offers a distinctive insight into the regulatory framework in Brazil. We explore the essential guidelines for quality testing, and nonclinical proof-of-concept considering the Brazilian Network of Experts in Advanced Therapies (RENETA) and International Standards and Guidelines (ICH e ISO). Adopting a multifaceted approach, our discussion incorporates scientific and industrial perspectives, addressing quality, biosafety, non-clinical viability, clinical trial and real-word data for pharmacovigilance demands. This comprehensive analysis presents a panoramic view of the development of skin TEPs, offering insights into the evolving landscape of this dynamic and promising field.

TRPA1 receptor modulation attenuates bladder overactivity induced by spinal cord injury.

The ankyrin-repeat transient receptor potential 1 (TRPA1) has been implicated in pathological conditions of the bladder, but its role in overactive bladder (OAB) following spinal cord injury (SCI) remains unknown. In this study, using a rat SCI model, we assessed the relevance of TRPA1 in OAB induced by SCI. SCI resulted in tissue damage, inflammation, and changes in bladder contractility and in voiding behavior. Moreover, SCI caused upregulation of TRPA1 protein and mRNA levels, in bladder and in dorsal root ganglion (DRG; L6-S1), but not in corresponding segment of spinal cord. Alteration in bladder contractility following SCI was evidenced by enhancement in cinnamaldehyde-, capsaicin-, or carbachol-induced bladder contraction as well as in its spontaneous phasic activity. Of relevance to voiding behavior, SCI induced increase in the number of nonvoiding contractions (NVCs), an important parameter associated with the OAB etiology, besides alterations in other urodynamic parameters. HC-030031 (TRPA1 antagonist) treatment decreased the number and the amplitude of NVCs while the TRPA1 antisense oligodeoxynucleotide (AS-ODN) treatment normalized the spontaneous phasic activity, decreased the cinnamaldehyde-induced bladder contraction and the number of NVCs in SCI rats. In addition, the cinnamaldehyde-induced bladder contraction was reduced by exposure of the bladder preparations to HC-030031. The efficacy of TRPA1 AS-ODN treatment was confirmed by means of the reduction of TRPA1 expression in the DRG, in the corresponding segment of the spinal cord and in the bladder, specifically in detrusor muscle. The present data show that the TRPA1 activation and upregulation seem to exert an important role in OAB following SCI.

The role of the paratrigeminal nucleus in the pressor response to sciatic nerve stimulation in the rat.

The paratrigeminal nucleus (Pa5), an input site for spinal, trigeminal, vagus and glossopharyngeal afferents, is a recognized site for orofacial nociceptive sensory processing. It has efferent connections to brain structures associated with nociception and cardiorespiratory functions. This study aimed at determining the function of the Pa5 on the cardiovascular component of the somatosensory reflex (SSR) to sciatic nerve stimulation (SNS) in paralyzed and artificially-ventilated rats following Pa5 chemical lesions (ibotenic acid), synaptic transmission blockade (CoCl(2)), local anaesthetics (lidocaine) or desensitization of primary afferent fibers (capsaicin). The pressor response to sciatic nerve stimulation at 0.6 mA and 20 Hz (14+/-1 mm Hg) was strongly attenuated by contra- (-80%) or bilateral (-50%) paratrigeminal nucleus lesions. Ipsilateral Pa5 lesions only attenuated the response to 0.1 mA, 20 Hz SNS (-55%). Cobalt chloride or lidocaine injected in the contralateral paratrigeminal nucleus also attenuated the SSR. In capsaicin-treated animals, the pressor responses to 0.1 mA were abolished, whereas the responses to SNS at 0.6 mA were increased from 65 to 100% depending on the stimulus frequency. The paratrigeminal nucleus receives both, excitatory and inhibitory components; the later apparently involving capsaicin-sensitive fiber inputs mostly to the ipsilateral site whereas the capsaicin insensitive excitatory components that respond to high or low frequency stimulation, respectively, target the contralateral and ipsilateral sites. Thus, the paratrigeminal nucleus mediates excitatory and inhibitory components of the somatosensory reflex, representing a primary synapse site in the brain for nociceptive inputs from the sciatic innervation field.

Sensory sciatic nerve afferent inputs to the dorsal lateral medulla in the rat.

Investigations show the paratrigeminal nucleus (Pa5) as an input site for sensory information from the sciatic nerve field. Functional or physical disruption of the Pa5 alters behavioral and somatosensory responses to nociceptive hindpaw stimulation or sciatic nerve electrostimulation (SNS), both contralateral to the affected structure. The nucleus, an input site for cranial and spinal nerves, known for orofacial nociceptive sensory processing, has efferent connections to structures associated with nociception and cardiorespiratory functions. This study aimed at determining the afferent sciatic pathway to dorsal lateral medulla by means of a neuronal tract-tracer (biocytin) injected in the iliac segment of the sciatic nerve. Spinal cord samples revealed bilateral labeling in the gracile and pyramidal or cuneate tracts from survival day 2 (lumbar L1/L2) to day 8 (cervical C2/C3 segments) following biocytin application. From day 10 to day 20 medulla samples showed labeling of the contralateral Pa5 to the injection site. The ipsilateral paratrigeminal nucleus showed labeling on day 10 only. The lateral reticular nucleus (LRt) showed fluorescent labeled terminal fibers on day 12 and 14, after tracer injection to contralateral sciatic nerve. Neurotracer injection into the LRt of sciatic nerve-biocytin-treated rats produced retrograde labeled neurons soma in the Pa5 in the vicinity of biocytin labeled nerve terminals. Therefore, Pa5 may be considered one of the first sites in the brain for sensory/nociceptive inputs from the sciatic nerve. Also, the findings include Pa5 and LRt in the neural pathway of the somatosympathetic pressor response to SNS and nocifensive responses to hindpaw stimulation.

Inhibition of spinal c-Jun-NH2-terminal kinase (JNK) improves locomotor activity of spinal cord injured rats.

Mitogen-activated protein kinases (MAPKs) have been implicated in central nervous system injuries, yet the roles within neurodegeneration following spinal cord injury (SCI) still remain partially elucidated. We aimed to investigate the changes in expression of the three MAPKs following SCI and the role of spinal c-jun-NH2-terminal kinase (JNK) in motor impairment following the lesion. SCI induced at the T9 level resulted in enhanced expression of phosphorylated MAPKs shortly after trauma. SCI increased spinal cord myeloperoxidase levels, indicating a local neutrophil infiltration, and elevated the number of spinal apoptotic cells. Intrathecal administration of a specific inhibitor of JNK phosphorylation, SP600125, given at 1 and 4h after SCI, reduced the p-JNK expression, the number of spinal apoptotic cells and many of the histological signs of spinal injury. Notably, restoration of locomotor performance was clearly ameliorated by SP600125 treatment. Altogether, the results demonstrate that SCI induces activation of spinal MAPKs and that JNK plays a major role in mediating the deleterious consequences of spinal injury, not only at the spinal level, but also those regarding locomotor function. Therefore, inhibition of JNK activation in the spinal cord shortly after trauma might constitute a feasible therapeutic strategy for the functional recovery from SCI.

Kinin and opioid receptors in the paratrigeminal nucleus modulate the somatosensory reflex to rat sciatic nerve stimulation.

The influence of kinin and opioid receptor blockade in the paratrigeminal nucleus (Pa5) on the somatosensory reflex (SSR) to sciatic nerve stimulation (SNS) was assessed in anaesthetized-paralyzed rats. SNS (square 1 ms pulses at 0.6 mA and 20 Hz for 10s) increased mean arterial pressure from 87+/-3 to 106+/-3 mmHg. Pressor responses to SNS were reduced 40-60% by HOE-140 and LF 16-0687 (B2 receptor antagonists; 20 and 100 pmol respectively), CTOP or nor-binaltorphimine (mu and kappa opioid receptor antagonists, respectively; 1 microg) but potentiated by naltrindole (delta opioid receptor antagonist) receptor antagonist microinjections into the contralateral (but not ipsilateral) Pa5. The SSR to sciatic nerve stimulation was not changed by B1 kinin receptor or NK1, NK2 and NK3 tachykinin receptor antagonists administered to the Pa5. Capsaicin pretreatment (40 mg/kg/day, 3 days) abolished the effects of the opioid receptor antagonists, but did not change the effect of kinin B2 receptor blockade on the SSR. Thus, the activity of B2 and opioid receptor-operated mechanisms in the Pa5 contribute to the SSR in the rat, suggesting a role for these endogenous peptides in the cardiovascular responses to SNS.

Influence of indomethacin on effects of endothelin-1 on guinea pig isolated rings of common bile duct and sphincter of Oddi.

The effects of endothelin-1 on motility of guinea pig extra-hepatic biliary tract portions were studied. Endothelin-1 (< or =100 nM) failed to contract rings of hepatic, cystic, proximal or distal common bile ducts, or choledochal or papillary halves of sphincter of Oddi. At 100 nM, endothelin-1 or sarafotoxin S6c (selective endothelin ET(B) receptor agonist) inhibited contractions of choledochal (but not papillary) sphincter of Oddi to carbachol (1 microM) by 63+/-5 and 45+/-9%, respectively. In distal common bile duct, indomethacin (5.6 microM) unmasked potent contractile effects of endothelin-1 [EC(50) 7.8 (5.5-11.1) nM; E(MAX) 80+/-6% of response to 80 mM KCl] and enhanced the contractile potency of carbachol (585-fold at EC(50) level), but not cholecystokinin C-terminal octapeptide. Inhibition of cholinergic responsiveness of the choledochal sphincter of Oddi by endothelin-1 was reduced by BQ-123 (1 microM; endothelin ET(A) receptor antagonist; cyclo[DTrp-DAsp-Pro-DVal-Leu]) and abolished by either BQ-123 plus BQ-788 (1 microM; endothelin ET(B) receptor antagonist; N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma-methylleucyl-D-1-methoxycarboyl-D-norleucine) or indomethacin. Thus, eicosanoids of the cyclo-oxygenase pathway (i.e. prostanoids) suppress endothelin-1-induced contractions of distal common bile duct and mediate endothelin ET(A) and ET(B) receptor-dependent inhibition of cholinergic responsiveness of the choledochal portion of the sphincter of Oddi.

Neuroprotective effect of the proanthocyanidin-rich fraction in experimental model of spinal cord injury.

OBJECTIVES: In this study, we evaluated the effect of the proanthocyanidins-rich fraction (PRF) obtained from Croton celtidifolius bark in an experimental animal model of spinal cord injury and cell death induced by glutamate. METHODS: Experiments were conducted using adult male Wistar rats (10 weeks old and weighing 270-300g). Experimental groups were randomly allocated into the following groups: spinal cord injury (SCI) + vehicle group: rats were subjected to SCI plus intraperitoneal administration of vehicle (saline 10 ml/kg); SCI + PRF: rats were subjected to SCI plus intraperitoneal administration of PRF (10 mg/kg) at 1 and 6 h after injury and sham operated. KEY FINDINGS: The treatment with the proanthocyanidin-rich fraction significantly improved not only motor recovery and grip force but also H2 O2 or glutamate-induced cell death and reactive oxygen species generation induced by glutamate in dorsal root ganglion cells. In this study we demonstrate that the neuroprotective effect triggered by the proanthocyanidins-rich fraction appears to be mediated in part by the inhibition of N-methyl-D-aspartate-type glutamate receptors. CONCLUSIONS: Taken together, our results demonstrate that PRF treatment ameliorates spinal cord injury and glutamatergic excitotoxicity and could have a potential therapeutic use.

Effects of kinin B(1) and B(2) receptor antagonists on overactive urinary bladder syndrome induced by spinal cord injury in rats.

BACKGROUND AND PURPOSE: Kinin B(1) and B(2) receptors have been implicated in physiological and pathological conditions of the urinary bladder. However, their role in overactive urinary bladder (OAB) syndrome following spinal cord injury (SCI) remains elusive. EXPERIMENTAL APPROACH: We investigated the role of kinin B(1) and B(2) receptors in OAB after SCI in rats. KEY RESULTS: SCI was associated with a marked inflammatory response and functional changes in the urinary bladder. SCI resulted in an up-regulation of B(1) receptor mRNA in the urinary bladder, dorsal root ganglion and spinal cord, as well as in B(1) protein in the urinary bladder and B(1) and B(2) receptor protein in spinal cord. Interestingly, both B(1) and B(2) protein expression were similarly distributed in detrusor muscle and urothelium of animals with SCI. In vitro stimulation of urinary bladder with the selective B(1) or B(2) agonist elicited a higher concentration-response curve in the SCI urinary bladder than in naive or sham urinary bladders. Cystometry revealed that treatment of SCI animals with the B(2) selective antagonist icatibant reduced the amplitude and number of non-voiding contractions (NVCs). The B(1) antagonist des-Arg(9) -[Leu(8) ]-bradykinin reduced the number of NVCs while the non-peptide B(1) antagonist SSR240612 reduced the number of NVCs, the urinary bladder capacity and increased the voiding efficiency and voided volume. CONCLUSIONS AND IMPLICATIONS: Taken together, these data show the important roles of B(1) and B(2) receptors in OAB following SCI in rats and suggest that blockade of these receptors could be a potential therapeutic target for controlling OAB.

[The blood-brain barrier and drug delivery in the central nervous system]. / La barrera hematoencefalica y la administracion de medicamentos en el sistema nervioso central.

AIM: To provide an updated view of the difficulties due to barriers and strategies used to allow the release of drugs in the central nervous system. INTRODUCTION: The difficulty for the treatment of many diseases of the central nervous system, through the use of intra-venous drugs, is due to the presence of barriers that prevent the release of the same: the blood-brain barrier, blood-cerebro-spinal fluid barrier and the blood-arachnoid barrier. DEVELOPMENT: The blood-brain barrier is the main barrier for the transport of drugs in the brain that also acts as a immunologic and metabolic barrier. The endothelial cells of the blood-brain barrier are connected to a junction complex through the interaction of transmembrane proteins that protrude from de inside to the outside, forming a connection between the endothelial cells. The transport of substances to the brain depends on the mechanisms of transport present in the barrier and the diffusion of these compounds also depends on the physicochemical characteristics of the molecule. Some diseases alter the permeability of the blood-brain barrier and thus the passage of drugs. Strategies such as the use of methods for drug delivery in the brain have been investigated. CONCLUSIONS: Further details regarding the mechanisms of transport across the blood-brain barrier and the changes in neuropathology would provide important information about the etiology of diseases and lead to better therapeutic strategies.

Role of the paratrigeminal nucleus in nocifensive responses of rats to chemical, thermal and mechanical stimuli applied to the hind paw.

Anatomical and immunohystochemical data suggest the paratrigeminal nucleus (Pa5) may play a role in nociceptive processing. The current study examines the influence of unilateral Pa5 lesion on nocifensive responses of conscious rats to noxious thermal (Hargreaves test), mechanical (electronic von Frey and Randall-Selitto tests), and chemical (formalin 2.5%; 50 microl) stimuli applied to the hind paw. Lesion of the Pa5 induced by ibotenic acid did not affect the latency for radiant heat-induced withdrawal of either paw. In contrast, the mean mechanical threshold for withdrawal of the contralateral (but not ipsilateral) paw in Pa5-lesioned rats was reduced by approximately 45% and 20%, in electronic von Frey and Randall-Selitto tests, respectively, when compared to sham-operated animals. Conversely, animals with Pa5 lesions injected with formalin in the contralateral paw spent less time engaged in focused (licking, biting or scratching the injected paw) and total nocifensive behavior (i.e., focused nocifensive behavior plus protection of the injected paw during movements) in both the first and second phases of the test [ approximately 50% inhibition of each parameter during first phase (0-5 min) and at 20, 25, and 30 min of second phase, relative to the sham-operated group], but the number of paw-jerks was unaffected. Pa5 lesion also delayed the onset of second phase focused pain induced by formalin in the ipsilateral paw. The results suggest that the Pa5 integrates the supraspinal pain control system and plays a differential modulatory role in the central processing of mechanical and chemical nociceptive information.

La barrera hematoencefálica y la administración de medicamentos en el sistema nervioso central / The blood-brain barrier and drug delivery in the central nervous system

Objetivo. Ofrecer una visión actualizada de las dificultades debido a las barreras y estrategias usadas para permitir la liberación de medicamentos en el sistema nervioso central. Introducción. La dificultad en el tratamiento de muchas enfermedades del sistema nervioso central, mediante el uso de fármacos por vía intravenosa, se debe a la presencia de barreras que impiden la liberación de éstos: la barrera hematoencefálica, la barrera sangre-líquido cefalorraquídeo y la barrera sangre-aracnoides. Desarrollo. La barrera hematoencefálica es la principal barrera para el transporte de medicamentos en el cerebro, que actúa también como una barrera inmunológica y metabólica. Las células endoteliales de la barrera hematoencefálica están conectadas a un complejo de uniones mediante la interacción de proteínas transmembranales que sobresalen del interior hacia el exterior, formando una conexión entre las células endoteliales. El transporte de sustancias al cerebro depende de los mecanismos de transporte presentes en la barrera y la difusión de estos compuestos depende también de las características fisicoquímicas de la molécula. Algunas enfermedades alteran la permeabilidad de la barrera hematoencefálica y, por lo tanto, el paso de los medicamentos. Se han investigado diferentes estrategias como métodos para la administración de medicamentos. Conclusiones. La obtención de un conocimiento más profundo de los mecanismos de transporte a través de la barrera hematoencefálica y los cambios en la neuropatología proporcionarían una información importante sobre la etiología de algunas enfermedades y conduciría a mejores estrategias terapéuticas (AU)

Aim. To provide an updated view of the difficulties due to barriers and strategies used to allow the release of drugs in the entral nervous system. Introduction. The difficulty for the treatment of many diseases of the central nervous system, through the use of intravenous drugs, is due to the presence of barriers that prevent the release of the same: the blood-brain barrier, bloodcerebrospinal fluid barrier and the blood-arachnoid barrier. Development. The blood-brain barrier is the main barrier for the transport of drugs in the brain that also acts as a immunologic and metabolic barrier. The endothelial cells of the blood-brain barrier are connected to a junction complex through the interaction of transmembrane proteins that protrude from de inside to the outside, forming a connection between the endothelial cells. The transport of substances to the brain depends on the mechanisms of transport present in the barrier and the diffusion of these compounds also depends on the physicochemical characteristics of the molecule. Some diseases alter the permeability of the blood-brain barrier and thus the passage of drugs. Strategies such as the use of methods for drug delivery in the brain have been investigated. Conclusions. Further details regarding the mechanisms of transport across the blood-brain barrier and the changes in neuropathology would provide important information about the etiology of diseases and lead to better therapeutic strategies (AU)