GABAergic neurons differentiated from BDNF- and Dlx2-modified neural stem cells restore disrupted neural circuits in brainstem stroke.

BACKGROUND: Brainstem stroke causes severe and persistent neurological impairment. Due to the limited spontaneous recovery and regeneration of the disrupted neural circuits, transplantation of exogenous neural stem cells (NSCs) was an alternative, while there were limitations for primitive NSCs. METHODS: We established a mouse model of brainstem stroke by injecting endothelin in the right pons. Brain-derived neurotrophic factor (BDNF)- and distal-less homeobox 2 (Dlx2)-modified NSCs were transplanted to treat brainstem stroke. Transsynaptic viral tracking, immunostaining, magnetic resonance imaging, behavioral testing, and whole-cell patch clamp recordings were applied to probe the pathophysiology and therapeutic prospects of BDNF- and Dlx2-modified NSCs. RESULTS: GABAergic neurons were predominantly lost after the brainstem stroke. No endogenous NSCs were generated in situ or migrated from the neurogenesis niches within the brainstem infarct region. Co-overexpressions of BDNF and Dlx2 not only promoted the survival of NSCs, but also boosted the differentiation of NSCs into GABAergic neurons. Results from transsynaptic virus tracking, immunostaining, and evidence from whole-cell patch clamping revealed the morphological and functional integration of the grafted BDNF- and Dlx2-modified NSCs-derived neurons with the host neural circuits. Neurological function was improved by transplantation of BDNF- and Dlx2-modified NSCs in brainstem stroke. CONCLUSIONS: These findings demonstrated that BDNF- and Dlx2-modified NSCs differentiated into GABAergic neurons, integrated into and reconstituted the host neural networks, and alleviated the ischemic injury. It thus provided a potential therapeutic strategy for brainstem stroke.

Heterogeneous pattern of gene expression driven by TTN mutation is involved in the construction of a prognosis model of lung squamous cell carcinoma.

Objective: To investigate the impact that TTN mutation had on the gene heterogeneity expression and prognosis in patients with lung adenocarcinoma. Methods: In this study, the Cancer Genome Atlas (TCGA) dataset was used to analyze the TTN mutations in lung adenocarcinoma. Lung adenocarcinoma data was collected from the TCGA database, clinical information of patients was analyzed, and bioinformatics statistical methods were applied for mutation analysis and prognosis survival analysis. The results were verified using the GEO dataset. Results: The incidence of TTN mutations in lung adenocarcinoma was found to be 73%, and it was related to the prognosis of lung adenocarcinoma. Ten genes were screened with significant contributions to prognosis. A prognosis model was constructed and verified by LASSO COX analysis in the TCGA and GEO datasets based on these ten beneficial factors. The independent prognostic factor H2BC9 for TTN mutation-driven gene heterogeneity expression was screened through multi-factor COX regression analysis. Conclusion: Our data showed that the gene heterogeneity expression, which was driven by TTN mutations, prolonged the survival of lung adenocarcinoma patients and provided valuable clues for the prognosis of TTN gene mutations in lung adenocarcinoma.

Inhibiting Cyclin B1-treated Pontine Infarction by Suppressing Proliferation of SPP1+ Microglia.

Pontine infarction is the major subtype of brainstem stroke causing severe neurological deficits. The pathophysiology and treatment of pontine infarction was rarely studied. A rat model of acute pontine infarction was established via injection of endothelin-1 in the pons. Single-cell RNA sequencing was applied to detect the cellular response in pontine infarction. Based on this finding, a potential treatment for pontine infarction targeting microglia was verified. Occlusion of penetrating artery caused by endothelin-1 led to pontine infarction. Single-cell RNA sequencing revealed a subtype of activated microglia, SPP1+ microglia, which were different from M1-like or M2-like depolarization. SPP1+ microglia interacted with oligodendrocytes and contributed to the demyelination of nerve tracts. Cyclin B1 regulated the proliferation of SPP1+ microglia. Cucurbitacin E, a cyclin B1 inhibitor, reduced the proliferation of SPP1+ microglia around the injured myelin sheath and alleviated the demyelination. Moreover, cucurbitacin E treatment decreased the ischemic infarction volume and neurological deficits after pontine infarction. SPP1+ microglia contributed to axonal demyelination in the pontine infarction, and inhibition of SPP1+ microglia provided neuroprotection for pontine infarction.

Massive Pontine Hemorrhage by Dual Injection of Autologous Blood.

We provide a protocol to establish a massive pontine hemorrhage model in a rat. Rats weighing about 250 grams were used in this study. One hundred microliters of autologous blood was taken from the tail vein and stereotaxically injected into the pons. The injection process was divided into 2 steps: First, 10 µL of blood was injected into a specific location, anteroposterior position (AP) -9.0 mm; lateral (Lat) 0 mm; vertical (Vert) -9.2 mm, followed by a second injection of the residual blood located at AP -9.0 mm; Lat 0 mm; Vert -9.0 mm with a 20-minute interval. The balance beam test, limb placement test, and the modified Voestch neuroscore were used to evaluate neurological function. Magnetic Resonance Imaging (MRI) was used to assess the volume of hemorrhage in vivo. The symptoms of this model were in line with patients with massive pontine hemorrhage.

Establishment of Acute Pontine Infarction in Rats by Electrical Stimulation.

Pontine infarction is the most common stroke subtype in the posterior circulation, while there lacks a rodent model mimicking pontine infarction. Provided here is a protocol for successfully establishing a rat model of acute pontine infarction. Rats weighing about 250 g are used, and a probe with an insulated sheath is injected into the pons using a stereotaxic apparatus. A lesion is produced by the electrical stimulation with a single pulse. The Longa score, Berderson score, and beam balance test are used to assess neurological deficits. Additionally, the adhesive-removal somatosensory test is used to determine sensorimotor function, and the limb placement test is used to evaluate proprioception. MRI scans are then used to assess the infarction in vivo, and TTC staining is used to confirm the infarction in vitro. Here, a successful infarction is identified that is located in the anterolateral basis of the rostral pons. In conclusion, a new method is described to establish an acute pontine infarction rat model.