Direct spinal ventral root repair following avulsion: effectiveness of a new heterologous fibrin sealant on motoneuron survival and regeneration

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOCAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIORAxonal injuries at the interface between central and peripheral nervous system, such as ventral root avulsion (VRA), induce important degenerative processes, mostly resulting in neuronal and motor function loss. In the present work, we have compared two different fibrin sealants, one derived from human blood and another derived from animal blood and Crotalus durissus terrificus venom, as a promising treatment for this type of injury. Lewis rats were submitted to VRA (L4-L6) and had the avulsed roots reimplanted to the surface of the spinal cord, with the aid of fibrin sealant. The spinal cords were processed to evaluate neuronal survival, synaptic stability, and glial reactivity, 4 and 12 weeks after lesion. Sciatic nerves were processed to investigate Schwann cell activity by p75(NTR) expression (4 weeks after surgery) and to count myelinated axons and morphometric evaluation (12 weeks after surgery). Walking track test was used to evaluate gait recovery, up to 12 weeks. The results indicate that both fibrin sealants are similarly efficient. However, the snake-derived fibrin glue is a potentially safer alternative for being a biological and biodegradable product which does not contain human blood derivatives. Therefore, the venom glue can be a useful tool for the scientific community due to its advantages and variety of applications.Axonal injuries at the interface between central and peripheral nervous system, such as ventral root avulsion (VRA), induce important degenerative processes, mostly resulting in neuronal and motor function loss. In the present work, we have compared two d2016116FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOCAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIORFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOCAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR2013/04409-02014/06892-3; 300552/2013-9; 310207/2011-8563582/2010-323038.000823/2011-21; 23038.005536/2012-3

Zika Virus Infection Causes Temporary Paralysis in Adult Mice With Motor Neuron Synaptic Retraction and Evidence for Proximal Peripheral Neuropathy

Clinical evidence is mounting that Zika virus can contribute to Guillain-Barré syndrome which causes temporary paralysis, yet the mechanism is unknown. We investigated the mechanism of temporary acute flaccid paralysis caused by Zika virus infection in aged interferon αβ-receptor knockout mice used for their susceptibility to infection. Twenty-five to thirty-five percent of mice infected subcutaneously with Zika virus developed motor deficits including acute flaccid paralysis that peaked 8-10 days after viral challenge. These mice recovered within a week. Despite Zika virus infection in the spinal cord, motor neurons were not destroyed. We examined ultrastructures of motor neurons and synapses by transmission electron microscopy. The percent coverage of motor neurons by boutons was reduced by 20%; more specifically, flattened-vesicle boutons were reduced by 46%, and were normalized in recovering mice. Using electromyographic procedures employed in people to help diagnose Guillain-Barré syndrome, we determined that nerve conduction velocities between the sciatic notch and the gastrocnemius muscle were unchanged in paralyzed mice. However, F-wave latencies were increased in paralyzed mice, which suggests that neuropathy may exist between the sciatic notch to the nerve rootlets. Reversible synaptic retraction may be a previously unrecognized cofactor along with peripheral neuropathy for the development of Guillain-Barré syndrome during Zika virus outbreaks

Anti-RBD IgG antibodies from endemic coronaviruses do not protect against the acquisition of SARS-CoV-2 infection among exposed uninfected individuals

BackgroundThe Coronaviridae family comprises seven viruses known to infect humans, classified into alphacoronaviruses (HCoV-229E and HCoV-NL63) and betacoronaviruses (HCoV-OC43 and HCoV-HKU1), which are considered endemic. Additionally, it includes SARS-CoV (severe acute respiratory syndrome), MERS-CoV (Middle East respiratory syndrome), and the novel coronavirus SARS-CoV-2, responsible for COVID-19. SARS-CoV-2 induces severe respiratory complications, particularly in the elderly, immunocompromised individuals and those with underlying diseases. An essential question since the onset of the COVID-19 pandemic has been to determine whether prior exposure to seasonal coronaviruses influences immunity or protection against SARS-CoV-2.MethodsIn this study, we investigated a cohort of 47 couples (N=94), where one partner tested positive for SARS-CoV-2 infection via real-time PCR while the other remained negative. Plasma samples, collected at least 30 days post-PCR reaction, were assessed using indirect ELISA and competition assays to measure specific antibodies against the receptor-binding domain (RBD) portion of the Spike (S) protein from SARS-CoV-2, HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKU1.ResultsIgG antibody levels against the four endemic coronavirus RBD proteins were similar between the PCR-positive and PCR-negative individuals, suggesting that IgG against endemic coronavirus RBD regions was not associated with protection from infection. Moreover, we found no significant IgG antibody cross-reactivity between endemic coronaviruses and SARS-CoV-2 RBDs.ConclusionsTaken together, results suggest that anti-RBD antibodies induced by a previous infection with endemic HCoVs do not protect against acquisition of COVID-19 among exposed uninfected individuals

Combination of heterologous fibrin sealant and bioengineered human embryonic stem cells to improve regeneration following autogenous sciatic nerve grafting repair

Abstract Background Peripheral nerve injury is a worldwide clinical problem, and the preferred surgical method for treating it is the end-to-end neurorrhaphy. When it is not possible due to a large nerve gap, autologous nerve grafting is used. However, these surgical techniques result in nerve regeneration at highly variable degrees. It is thus very important to seek complementary techniques to improve motor and sensory recovery. One promising approach could be cell therapy. Transplantation therapy with human embryonic stem cells (hESCs) is appealing because these cells are pluripotent and can differentiate into specialized cell types and have self-renewal ability. Therefore, the main objective of this study was to find conditions under which functional recovery is improved after sciatic nerve neurorrhaphy. We assumed that hESC, either alone or in combination with heterologous fibrin sealant scaffold, could be used to support regeneration in a mouse model of sciatic nerve injury and repair via autografting with end-to-end neurorrhaphy. Methods Five millimeters of the sciatic nerve of C57BL/6 J mice were transected off and rotated 180 degrees to simulate an injury, and then stumps were sutured. Next, we applied heterologous fibrin sealant and/or human embryonic stem cells genetically altered to overexpress fibroblast growth factor 2 (FGF2) at the site of the injury. The study was designed to include six experimental groups comprising neurorrhaphy (N), neurorrhaphy + heterologous fibrin sealant (N + F), neurorrhaphy + heterologous fibrin sealant + doxycycline (N + F + D), neurorrhaphy + heterologous fibrin sealant + wild-type hESC (N + F + W), neurorrhaphy + heterologous fibrin sealant + hESC off (N + F + T), and neurorrhaphy + heterologous fibrin sealant + hESC on via doxycycline (N + F + D + T). We evaluated the recovery rate using Catwalk and von Frey functional recovery tests, as well as immunohistochemistry analysis. Results The experiments indicated that sensory function improved when transgenic hESCs were used. The regeneration of sensory fibers indeed led to increased reflexes, upon stimulation of the paw ipsilateral to the lesion, as seen by von-Frey evaluation, which was supported by immunohistochemistry. Conclusions Overall, the present data demonstrated that transgenic embryonic stem cells, engineered to overexpress FGF-2 in an inducible fashion, could be employed to support regeneration aiming at the recovery of both motor and sensory functions

Spinal Reflex Recovery after Dorsal Rhizotomy and Repair with Platelet-Rich Plasma (PRP) Gel Combined with Bioengineered Human Embryonic Stem Cells (hESCs)

Dorsal root rhizotomy (DRZ) is currently considered an untreatable injury, resulting in the loss of sensitive function and usually leading to neuropathic pain. In this context, we recently proposed a new surgical approach to treat DRZ that uses platelet-rich plasma (PRP) gel to restore the spinal reflex. Success was correlated with the reentry of primary afferents into the spinal cord. Here, aiming to enhance previous results, cell therapy with bioengineered human embryonic stem cells (hESCs) to overexpress fibroblast growth factor 2 (FGF2) was combined with PRP. For these experiments, adult female rats were submitted to a unilateral rhizotomy of the lumbar spinal dorsal roots, which was followed by root repair with PRP gel with or without bioengineered hESCs. One week after DRZ, the spinal cords were processed to evaluate changes in the glial response (GFAP and Iba-1) and excitatory synaptic circuits (VGLUT1) by immunofluorescence. Eight weeks postsurgery, the lumbar intumescences were processed for analysis of the repaired microenvironment by transmission electron microscopy. Spinal reflex recovery was evaluated by the electronic Von Frey method for eight weeks. The transcript levels for human FGF2 were over 37-fold higher in the induced hESCs than in the noninduced and the wildtype counterparts. Altogether, the results indicate that the combination of hESCs with PRP gel promoted substantial and prominent axonal regeneration processes after DRZ. Thus, the repair of dorsal roots, if done appropriately, may be considered an approach to regain sensory-motor function after dorsal root axotomy

Reflex arc recovery after spinal cord dorsal root repair with platelet rich plasma (PRP)

Spinal dorsal roots can be affected by a wide range of lesions, leading to a significant loss of proprioceptive information transmission and greatly affecting motor behavior. In this context, the reimplantation of lesioned roots with platelet-rich plasma (PRP) may allow nerve regeneration. Therefore, the present study evaluated sensorimotor improvement following dorsal root rhizotomy and repair with PRP. For this purpose, female Lewis rats were subjected to unilateral rhizotomy (RZ) of the L4-L6 dorsal roots and divided into the following groups: (1) the unlesioned control group; (2) the group that underwent rhizotomy (RZ) without repair; and (3) the group that underwent RZ followed by root repair with PRP. PRP was obtained from human blood and characterized regarding platelet concentration, integrity, and viability. Reflex arc recovery was evaluated weekly for eight weeks by the electronic von Frey method. The spinal cords were processed 1 week postlesion to evaluate the in vivo gene expression of TNFa, TGF-beta, BDNF, GDNF, VEGF, NGF, IL-4, IL-6, IL-13 by qRT-PCR and eight weeks postlesion to evaluate changes in the glial response (GFAP and Iba-1) and excitatory synaptic circuits (VGLUT1) by immunofluorescence. The results indicated that PRP therapy partially restores the paw withdrawal reflex over time, indicating the reentry of primary afferents from the dorsal root ganglia into the spinal cord without exacerbating glial reactivity. Additionally, the analysis of mRNA levels showed that PRP therapy has immunomodulatory properties. Overall, the present data suggest that the repair of dorsal roots with PRP may be considered a promising approach to improve sensorimotor recovery following dorsal rhizotomy152212224CNPQ - Conselho Nacional de Desenvolvimento Científico e TecnológicoFAPESB – Fundação de Amparo á Pesquisa Do Estado Da Bahia303085/2017-72015/26206-0; 2014/06892-3; 2018/05006-

Development of a device useful to reproducibly produce large quantities of viable and uniform stem cell spheroids with controlled diameters

Three-dimensional cellular aggregates can mimic the natural microenvironment of tissues and organs and obtaining them through controlled and reproducible processes is mandatory for scaling up and implementing drug cytotoxicity and efficacy tests, as well as tissue engineering protocols. The purpose of this work was to develop and evaluate the performance of a device with two different geometries fabricated by additive manufacturing. The methodology was based on casting a microwell array insert using a non-adhesive hydrogel to obtain highly regular microcavities to standardize spheroid formation and morphology. Spheroids of dental pulp stem cells, bone marrow stromal cells and embryonic stem cells showing high cell viability and average diameters of around 253, 220, and 500 μm, respectively, were produced using the device with the geometry considered most adequate. The cell aggregates showed sphericity indexes above 0.9 and regular surfaces (solidity index higher than 0.96). Around 1000 spheroids could be produced in a standard six-well plate. Overall, these results show that this method facilitates obtaining a large number of uniform, viable spheroids with pre-specified average diameters and through a low-cost and reproducible process for a myriad of applications

Fibrin biopolymer as scaffold candidate to treat bone defects in rats

Bone tissue repair remains a challenge in tissue engineering. Currently, new materials are being applied and often integrated with live cells and biological scaffolds. The fibrin biopolymer (FBP) proposed in this study has hemostatic, sealant, adhesive, scaffolding and drug-delivery properties. The regenerative potential of an association of FBP, biphasic calcium phosphate (BCP) and mesenchymal stem cells (MSCs) was evaluated in defects of rat femurs. Methods: Adult male Wistar rats were submitted to a 5-mm defect in the femur. This was filled with the following materials and/or associations: BPC; FBP and BCP; FBP and MSCs; and BCP, FBP and MSCs. Bone defect without filling was defined as the control group. Thirty and sixty days after the procedure, animals were euthanatized and subjected to computed tomography, scanning electron microscopy and qualitative and quantitative histological analysis. Results: It was shown that FBP is a suitable scaffold for bone defects due to the formation of a stable clot that facilitates the handling and optimizes the surgical procedures, allowing also cell adhesion and proliferation. The association between the materials was biocompatible. Progressive deposition of bone matrix was higher in the group treated with FBP and MSCs. Differentiation of mesenchymal stem cells into osteogenic lineage was not necessary to stimulate bone formation. Conclusions: FBP proved to be an excellent scaffold candidate for bone repair therapies due to application ease and biocompatibility with synthetic calcium-based materials. The satisfactory results obtained by the association of FBP with MSCs may provide a more effective and less costly new approach for bone tissue engineering25CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPES563582/2010-3; 404770/2018-523038.000823/2011-21This project was supported by the Brazilian National Council for Scientific and Technological Development (CNPq), in partnership with the Ministry of Health of Brazil (Decit/SCTIE/MS), proc. n. 563582/2010-3 and the Coordination for the Improvement of Higher Education Personnel (CAPES), Toxinology AUX-PE-1219/2011, proc. n. 23038.000823/2011-21. Moreover, this publication was supported in part by CNPq and CAPES through “Programa Editorial CNPq/CAPES” call n. 18/2018, grant n. 404770/2018-