Clinical implications of single cell sequencing for bladder cancer.

Bladder cancer (BC) is the 10th most common cancer worldwide, with about 0.5 million reported new cases and about 0.2 million deaths per year. In this scoping review, we summarize the current evidence regarding the clinical implications of single-cell sequencing for bladder cancer based on PRISMA guidelines. We searched PubMed, CENTRAL, Embase, and supplemented with manual searches through the Scopus, and Web of Science for published studies until February 2023. We included original studies that used at least one single-cell technology to study bladder cancer. Forty-one publications were included in the review. Twenty-nine studies showed that this technology can identify cell subtypes in the tumor microenvironment that may predict prognosis or response to immune checkpoint inhibition therapy. Two studies were able to diagnose BC by identifying neoplastic cells through single-cell sequencing urine samples. The remaining studies were mainly a preclinical exploration of tumor microenvironment at single cell level. Single-cell sequencing technology can discriminate heterogeneity in bladder tumor cells and determine the key molecular properties that can lead to the discovery of novel perspectives on cancer management. This nascent tool can advance the early diagnosis, prognosis judgment, and targeted therapy of bladder cancer.

Pregabalin synchronizes the regeneration of nerve and muscle fibers optimizing the gait recovery of MDX dystrophic mice.

Duchenne muscular dystrophy (DMD) is an X-linked genetic disorder induced by mutations in the dystrophin gene, leading to a degeneration of muscle fibers, triggering retrograde immunomodulatory, and degenerative events in the central nervous system. Thus, neuroprotective drugs such as pregabalin (PGB) can improve motor function by modulating plasticity, together with anti-inflammatory effects. The present work aimed to study the effects of PGB on axonal regeneration after axotomy in dystrophic and non-dystrophic mice. For that, MDX and C57BL/10 mouse strains were subjected to peripheral nerve damage and were treated with PGB (30 mg/kg/day, i.p.) for 28 consecutive days. The treatment was carried out in mice as soon as they completed 5 weeks of life, 1 week before the lesion, corresponding to the peak period of muscle degeneration in the MDX strain. Six-week-old mice were submitted to unilateral sciatic nerve crush and were sacrificed in the 9th week of age. The ipsi and contralateral sciatic nerves were processed for immunohistochemistry and qRT-PCR, evaluating the expression of proteins and gene transcripts related to neuronal and Schwann cell activity. Cranial tibial muscles were dissected for evaluation of neuromuscular junctions using α-bungarotoxin, and the myelinated axons of the sciatic nerve were analyzed by morphometry. The recovery of motor function was monitored throughout the treatment through tests of forced locomotion (rotarod) and spontaneous walking track test (Catwalk system). The results show that treatment with PGB reduced the retrograde cyclic effects of muscle degeneration/regeneration on the nervous system. This fact was confirmed after peripheral nerve injury, showing better adaptation and response of neurons and glia for rapid axonal regeneration, with efficient muscle targeting and regain of function. No side effects of PGB treatment were observed, and the expression of pro-regenerative proteins in neurons and Schwann cells was upregulated. Morphometry of the axons was in line with the preservation of motor endplates, resulting in enhanced performance of dystrophic animals. Overall, the present data indicate that pregabalin is protective and enhances regeneration of the SNP during the development of DMD, improving motor function, which can, in turn, be translated to the clinic.

TNF-mimetic peptide mixed with fibrin glue improves peripheral nerve regeneration.

Surgical intervention is necessary following nerve trauma. Tubular prostheses can guide growing axons and inserting substances within these prostheses can be positive for the regeneration, making it an alternative for the current standard tools for nerve repair. Our aim was to investigate the effects of fibrin glue BthTL when combined with a synthetic TNF mimetic-action peptide on nerve regeneration. Male Wistar rats suffered left sciatic nerve transection. For repairing, we used empty silicon tubes (n = 10), tubes filled with fibrin glue BthTL (Tube + Glue group, n = 10) or tubes filled with fibrin glue BThTL mixed with TNF mimetic peptide (Tube + Glue + Pep group, n = 10). Animals were euthanized after 45 days. We collected nerves to perform immunostaining (neurofilament, GAP43, S100-ß, NGFRp75 and Iba-1), light and transmission electron microscopy (for counting myelinated, unmyelinated and degenerated fibers; and for the evaluation of morphometric aspects of regenerated fibers) and collagen staining. All procedures were approved by local ethics committee (protocol 063/17). Tube + Glue + Pep group showed intense inflammatory infiltrate, higher Iba-1 expression, increased immunostaining for NGFRp75 receptor (which characterizes Schwann cell regenerative phenotype), higher myelin thickness and fiber diameter and more type III collagen deposition. Tube + Glue group showed intermediate results between empty tube and Tube + Glue + Pep groups for anti-NGFRp75 immunostaining, inflammation and collagen; on fiber counts, this group showed more degenerate fibers and fewer unmyelinated axons than others. Empty tube group showed superiority only in GAP43 immunostaining. A combination of BthTL glue and TNF mimetic peptide induced greater axonal regrowth and remyelination.

Pregabalin-induced neuroprotection and gait improvement in dystrophic MDX mice.

Duchenne muscular dystrophy (DMD) is a genetic disease linked to the X chromosome induced by mutations in the dystrophin gene. Neuroprotective drugs, such as pregabalin (PGB), can improve motor function through the modulation of excitatory synapses, together with anti-apoptotic and anti-inflammatory effects. The present work studied the effects of PGB in the preservation of dystrophic peripheral nerves, allowing motor improvements in MDX mice. Five weeks old MDX and C57BL/10 mice were treated with PGB (30 mg/kg/day, i.p.) or vehicle, for 28 consecutive days. The mice were sacrificed on the 9th week, the sciatic nerves were dissected out and processed for immunohistochemistry and qRT-PCR, for evaluating the expression of proteins and gene transcripts related to neuronal activity and Schwann cell function. The lumbar spinal cords were also processed for qRT-PCR to evaluate the expression of neurotrophic factors and pro- and anti-inflammatory cytokines. Cranial tibial muscles were dissected out for endplate evaluation with α-bungarotoxin. The recovery of motor function was monitored throughout the treatment, using a spontaneous walking track test (Catwalk system) and a forced locomotion test (Rotarod). The results showed that treatment with PGB reduced the retrograde effects of muscle degeneration/regeneration on the nervous system from the 5th to the 9th week in MDX mice. Thus, PGB induced protein expression in neurons and Schwann cells, protecting myelinated fibers. In turn, better axonal morphology and close-to-normal motor endplates were observed. Indeed, such effects resulted in improved motor coordination of dystrophic animals. We believe that treatment with PGB improved the balance between excitatory and inhibitory inputs to spinal motoneurons, increasing motor control. In addition, PGB enhanced peripheral nerve homeostasis, by positively affecting Schwann cells. In general, the present results indicate that pregabalin is effective in protecting the PNS during the development of DMD, improving motor coordination, indicating possible translation to the clinic.

Angiotensin-(1-7) receptor Mas antagonist (A779) influenced gliosis and reduced synaptic density in the spinal cord after peripheral axotomy.

The peptide angiotensin-(1-7) [Ang (1-7)] and its receptor Mas are involved in controlling arterial pressure and display actions on the nervous system. In a previous study, our laboratory showed that A779 [(peptidyl antagonist of the Ang-(1-7)] treatment had a negative effect following a lesion of the sciatic nerve, possibly by delaying the responses of Schwann cells, resulting in a decreased axonal organization along with a slowed functional return. In the present work, we investigated the central cellular changes after sciatic nerve injury in rodents treated with A779 after two weeks. In the lumbar spinal cords, where the neuronal bodies that make up the sciatic are, the treatment with A779 showed reduced reactivity of astrocytes (p = 0.004, Mann-Whitney U test) and less synaptic density (p = 0.004, Mann-Whitney U test) after injury. Also, the treatment upregulated microglia activity in both sides (p = 0.004, Mann-Whitney U test), ipsilateral and contralateral to the lesion, of the spinal cord. In addition, the Mas expression in spine neurons was increased in response to axotomy especially after two weeks (p = 0.03, Mann-Whitney U test) following the nerve lesion in comparison to earlier stages after injury. Therefore, we can conclude that Ang-(1-7)/Mas axis plays a role during spinal cord recovery after peripheral nerve injury.

Pattern of Mas expression in acute and post-acute stage of nerve injury in mice.

Angiotensin-(1-7) (Ang [1-7]) and its receptor Mas are involved in a number of physiological processes, including control of arterial pressure and modulation of nervous system actions. However, the involvement of the Ang-(1-7)/Mas axis in peripheral nerve injury has not been investigated. Using a model of sciatic nerve injury in mice, we demonstrated opposing changes in Mas receptor expression at days 2 and 14 post-injury. Mas receptor expression was more intense 2days after the nerve lesion, compared with the intensity of the intact nerve. At this time point, the sciatic nerve functional index was -20. At day 14 after the lesion, the intensity of the immunostaining labeling in longitudinal sections of the nerve was reduced (∼30%) and the functional index increased +36 (gait improvement). In the axotomized group treated with A779 (a Mas receptor antagonist), the functional recovery index decreased in relation to the untreated axotomized group. The Mas receptor inhibitor also altered the intensity of labeling of S-100, GAP43, and IBA-1 (morphological features compatible with delayed axon growth). This study demonstrated that Ang-(1-7)/Mas axis activity was differentially modulated in the acute and post-acute stages of nerve injury.