Next generation sequencing of RB1gene for the molecular diagnosis of ethnic minority with retinoblastoma in Yunnan.

BACKGROUND: Retinoblastoma is a rare intraocular malignancy and typically initiated by inactivating biallelic mutations of RB1 gene. Each year, ~ 8000 children worldwide are diagnosed for retinoblastoma. In high-income countries, patient survival is over 95% while low-income countries is ~ 30%.If disease is diagnosed early and treated in centers specializing in retinoblastoma, the survival might exceed 95% and many eyes could be safely treated and support a lifetime of good vision. In China, approximate 1100 newly diagnosed cases are expected annually and 28 hospitals covering 25 provinces established centers classified by expertise and resources for better treatment options and follow-up. Comparing with other province of eastern China, Yunnan province is remote geographically. This might result that healthcare staff have low awareness of the role of genetic testing in management and screening in families. METHODS: The patients with retinoblastoma were selected in Yunnan. DNA from blood was used for targeted gene sequencing. Then, an in-house bioinformatics pipeline was done to detect both single nucleotide variants and small insertions/deletions. The pathogenic mutations were identified and further confirmed by conventional methods and cosegregation in families. RESULTS: Using our approach, targeted next generation sequencing was used to detect the mutation of these 12 probands. Bioinformatic predictions showed that nine mutations were found in our study and four were novel pathogenic variants in these nine mutations. CONCLUSIONS: It's the first report to describe RB1 mutations in Yunnan children with retinoblastoma. This study would improve role of genetic testing for management and family screening.

Rifampicin attenuates experimental autoimmune encephalomyelitis by inhibiting pathogenic Th17 cells responses.

Rifampicin, a broad-spectrum antibiotic, has neuroprotective, immunosuppressive, and anti-inflammatory properties. However, the effect of rifampicin on autoimmune disorders of the nervous system is not clear. In this study, we investigated whether rifampicin was beneficial to myelin oligodendrocyte glycoprotein peptide (MOG33-35 )-induced female C57BL/6 experimental autoimmune encephalomyelitis (EAE) mice, the well-established animal model of multiple sclerosis. Rifampicin treatment (daily from the first day after EAE immunization) remarkably attenuated clinical signs and loss of body weight, which are associated with suppression of inflammatory infiltration and demyelination in spinal cords of EAE mice. Furthermore, rifampicin dramatically reduced the disruption of blood-brain barrier integrity, down-regulated serum concentration of IL-6 and IL-17A, inhibited pathological Th17 cell differentiation, and modulated the expression of p-STAT3 and p-p65. These results suggest that rifampicin is effective for attenuating the clinical severity of EAE mice, which may be related to its inhibitive ability in differentiation of Th17 cell and secretion of its key effector molecule IL-17A via regulation of excessive activation of the key signaling molecules of JAK/STAT pathway. Our findings may be helpful for developing therapeutic and preventive strategies for multiple sclerosis.

A Modular Assembly of Spinal Cord-Like Tissue Allows Targeted Tissue Repair in the Transected Spinal Cord.

Tissue engineering-based neural construction holds promise in providing organoids with defined differentiation and therapeutic potentials. Here, a bioengineered transplantable spinal cord-like tissue (SCLT) is assembled in vitro by simulating the white matter and gray matter composition of the spinal cord using neural stem cell-based tissue engineering technique. Whether the organoid would execute targeted repair in injured spinal cord is evaluated. The integrated SCLT, assembled by white matter-like tissue (WMLT) module and gray matter-like tissue (GMLT) module, shares architectural, phenotypic, and functional similarities to the adult rat spinal cord. Organotypic coculturing with the dorsal root ganglion or muscle cells shows that the SCLT embraces spinal cord organogenesis potentials to establish connections with the targets, respectively. Transplantation of the SCLT into the transected spinal cord results in a significant motor function recovery of the paralyzed hind limbs in rats. Additionally, targeted spinal cord tissue repair is achieved by the modular design of SCLT, as evidenced by an increased remyelination in the WMLT area and an enlarged innervation in the GMLT area. More importantly, the pro-regeneration milieu facilitates the formation of a neuronal relay by the donor neurons, allowing the conduction of descending and ascending neural inputs.

Integration of donor mesenchymal stem cell-derived neuron-like cells into host neural network after rat spinal cord transection.

Functional deficits following spinal cord injury (SCI) primarily attribute to loss of neural connectivity. We therefore tested if novel tissue engineering approaches could enable neural network repair that facilitates functional recovery after spinal cord transection (SCT). Rat bone marrow-derived mesenchymal stem cells (MSCs), genetically engineered to overexpress TrkC, receptor of neurotrophin-3 (NT-3), were pre-differentiated into cells carrying neuronal features via co-culture with NT-3 overproducing Schwann cells in 3-dimensional gelatin sponge (GS) scaffold for 14 days in vitro. Intra-GS formation of MSC assemblies emulating neural network (MSC-GS) were verified morphologically via electron microscopy (EM) and functionally by whole-cell patch clamp recording of spontaneous post-synaptic currents. The differentiated MSCs still partially maintained prototypic property with the expression of some mesodermal cytokines. MSC-GS or GS was then grafted acutely into a 2 mm-wide transection gap in the T9-T10 spinal cord segments of adult rats. Eight weeks later, hindlimb function of the MSC-GS-treated SCT rats was significantly improved relative to controls receiving the GS or lesion only as indicated by BBB score. The MSC-GS transplantation also significantly recovered cortical motor evoked potential (CMEP). Histologically, MSC-derived neuron-like cells maintained their synapse-like structures in vivo; they additionally formed similar connections with host neurites (i.e., mostly serotonergic fibers plus a few corticospinal axons; validated by double-labeled immuno-EM). Moreover, motor cortex electrical stimulation triggered c-fos expression in the grafted and lumbar spinal cord cells of the treated rats only. Our data suggest that MSC-derived neuron-like cells resulting from NT-3-TrkC-induced differentiation can partially integrate into transected spinal cord and this strategy should be further investigated for reconstructing disrupted neural circuits.

Postnatal development of nestin positive neurons in rat basal forebrain: different onset and topography with choline acetyltransferase and parvalbumin expression.

Our previous studies identified a sub-population of cholinergic neurons which express nestin in the rostral part of the basal forebrain (BF) in normal adult rats. In the present study, the postnatal developmental patterns of nestin, choline acetyl transferase (ChAT) and parvalbumin (PV) positive neurons were explored by means of immunohistochemistry combined with immunofluorescence double label methods. Compared with early onset of ChAT expression (from P1) and delayed onset of PV expression (from P16), nestin positive activity was detected in the BF from P9 and co-expressed by parts of the ChAT positive neurons within the same region during the whole postnatal development process. However, ChAT and PV were not coexpressed by the neurons within the medial septum-diagonal band of Broca (MS-DBB) of BF. These results might imply a composite of separate development patterns displayed by different subpopulations of cholinergic neurons (nestin positive cholinergic neurons and nestin negative cholinergic neurons) within this region. Moreover, the topographic distribution of nestin, ChAT and PV positive neurons also showed different characteristics. In summary, our present study revealed a remarkable timing and topographic difference on the postnatal development of the nestin expression within the MS-DBB of BF compared with ChAT and PV expression. It is further suggested that nestin is re-expressed by cholinergic neurons in the BF after differentiation but not persisted from neuronal precursor cells.

The integration of NSC-derived and host neural networks after rat spinal cord transection.

Rebuilding structures that can bridge the injury gap and enable signal connection remains a challenging issue in spinal cord injury. We sought to determine if genetically enhanced expression of TrkC in neural stem cells (NSCs) and neurotrophin-3 in Schwann cells (SCs) co-cultured in a gelatin sponge scaffold could constitute a neural network, and whether it could act as a relay to rebuilt signal connection after spinal cord transection. Indeed, many NSCs in the scaffold assumed neuronal features including formation of synapses. By whole-cell patch clamp, the synapses associated with NSC-derived neurons were excitable. Grafting of the scaffold with differentiating NSCs + SCs into rats with a segment of the spinal cord removed had resulted in a significant functional recovery of the paralyzed hind-limbs. Remarkably, the NSC-derived neurons formed new synaptic contacts suggesting that the scaffold can form a relay for conduction of signals through the injury gap of spinal cord.

Chemical identification of nestin-immunoreactive neurons in the rat basal forebrain: a re-examination.

This study explores recently identified neurons that express the protein nestin in the medial septum-diagonal band of Broca (MS-DBB) of adult rats and humans. These nestin positive neurons from MS-DBB are known to project to the hippocampus and frontal cortex of the brain. However, their chemical identification has not been fully elucidated. In this study, we further investigated the chemical identity of the nestin-immunoreactive (ir) neurons in rats using double immunofluorescence labeling and single cell reverse transcription polymerase chain reaction (RT-PCR) techniques. The results of double labeling showed that all nestin-ir neurons exhibited choline acetyltransferase (ChAT) immunoreactivity in the MS-DBB of the basal forebrain. Conversely, only about 43% of the ChAT-ir neurons showed nestin immunoreactivity. In addition, a vast majority of the nestin-ir neurons (95%) were nerve growth factor receptor (NGFR) positive. The nestin-ir neurons were highly distributed in the rostral and intermediated regions of the MS-DBB. Single cell RT-PCR results showed that 90% of the nestin mRNA expressed neurons displayed ChAT mRNA expression as well, but neither the mRNA of the proteins glutamic acid decarboxylases 67 (GAD67) nor vesicular glutamate transporters (VGLUTs). These results provide the first evidence that nestin-ir neurons in the basal forebrain are a subpopulation of the classic cholinergic neurons. With high NGFR proteins activated in the nestin-ir neurons, we propose that the presence of nestin in the cholinergic neurons might be related to the survival and plasticity of the cholinergic neurons.

Survival, regeneration and functional recovery of motoneurons after delayed reimplantation of avulsed spinal root in adult rat.

We have established that extensive reinnervation and functional recovery follow immediate reimplantation of avulsed ventral roots in adult rats. In the present study, we examined the consequences of reimplantation delayed for 2 weeks after avulsion of the C6 spinal root. Twelve and 20 weeks after delayed reimplantation, 57% and 53% of the motoneurons in the injured spinal segment survived. More than 80% of surviving motoneurons regenerated axons into the reimplanted spinal root. Cholinesterase-silver staining revealed axon terminals on endplates in the denervated muscles. The biceps muscles in reimplanted animals had atrophied less than those in animals with avulsion only, as indicated by muscle wet weight and histological appearance. After electrical stimulation of the motor cortex or the C6 spinal root, typical EMG signals were recorded in biceps of reimplanted animals. The latency of the muscle potential at 20 weeks was similar to that of sham-operated controls. Behavioral recovery was demonstrated by a grooming test and ipsilateral forepaw movements were well coordinated in both voluntary and automatic activities. These results demonstrate that ventral root reimplantation can protect severed motoneurons, enable the severed motoneurons to regenerate axons, and enhance the recovery of forelimb function even when it is delayed for 2 weeks after avulsion.

Survival, regeneration and functional recovery of motoneurons in adult rats by reimplantation of ventral root following spinal root avulsion.

We investigated the functional recovery of motoneurons after reimplanting an avulsed ventral root in a rat model of traction injury. The eighth cervical root (C8) was avulsed by controlled traction and immediately reimplanted to the spinal cord. Spinal nerves from neighbouring segments (C5, C6, C7 and T1) were ligated and cut. After 12 or 20 weeks, the survival, regeneration and functional recovery of spinal motoneurons were evaluated by Nissl staining, retrograde labelling of motoneurons, NOS histochemistry, histological examination of muscle and nerve-muscle junction, electromyography and behavioural observation. In the control animals, about 14% or 11% of spinal motoneurons survived 12 or 20 weeks postinjury, respectively. By contrast, in animals with ventral root reimplantation, 62% and 55% of motoneurons survived at 12 or 20 weeks postinjury, respectively. Retrograde labelling and histological examination indicated that about 90% of the surviving motoneurons in the C8 segment regenerated axons into the reimplanted ventral root. Staining the muscles with silver and cholinesterase revealed new motor endplates in the reinnervated muscle. Functionally significant electromyographic responses in flexor digitorum superficialis and flexor carpi radialis were observed in experimental animals; however, the average latency of the motor action potentials was greater than normal control. The grasping test showed functional recovery of finger flexors and median nerve. In conclusion, our results indicate that spinal motoneurons can regenerate axons through reimplanted roots and reinnervate muscles to recover partial function.