Gene transfer to human trabecular meshwork cells in vitro and ex vivo using HIV-based lentivirus

AIM: To investigate whether the enhanced green fluorescent protein(EGFP) reporter gene could be transferred into human trabecular meshwork(HTM) cells by a HIV-based lentivirus both in vitro and ex vivo.METHODS: The HIV-based lentivirus that contains an EF1-α promoter driving EGFP expression cassette was constructed following the standard molecular cloning methods. The cultured HTM cells were transduced at a range of multiplicity of infection(MOI) with HIV-based lentivirus. EGFP positive cell populations were detected by flow cytometry. Human anterior eye segments were cultured with perfusion culture system and transfected by HIV-based lentivirus with a 1 ×108transducing unit(TU) virus in perfusion liquid. The intraocular pressure was recorded every 8h for 21 d. The expression of EGFP in the anterior segment of the human eye was detected by fluorescence microscopy. Furthermore, the distribution of EGFP expression was confirmed by anti-EGFP immunohistochemical staining.RESULTS: The HIV-based lentivirus which contains an EF1-α promoter driving EGFP expression cassette was constructed successfully. After HTM cells were transduced with HIV-based lentivirus containing EGFP in vitro, the ratio of EGFP positive cells to the total cell number reached 92.3%, with the MOI of 15. After the lentivirus containing EGFP were used to transduce human anterior eye segments, the EGFP could be directly detected by fluorescence microscopy in vivo.Immunohistochemistry staining revealed that 88.19%EGFP-positive trabecular meshwork(TM) cells were observed in the human anterior segment. Nevertheless,the intraocular pressure in the lentivirus-transduced group kept constant when compared with control group(P 】0.05).CONCLUSION: EGFP gene could be efficiently transferred into HTM cells both in vitro and ex vivo by using HIV-based lentivirus.

Co-localization of two-color rAAV2-retro confirms the dispersion characteristics of efferent projections of mitral cells in mouse accessory olfactory bulb

The accessory olfactory bulb(AOB), located at the posterior dorsal aspect of the main olfactory bulb(MOB), is the first brain relay of the accessory olfactory system(AOS), which can parallelly detect and process volatile and nonvolatile social chemosignals and mediate different sexual and social behaviors with the main olfactory system(MOS). However, due to its anatomical location and absence of specific markers, there is a lack of research on the internal and external neural circuits of the AOB. This issue was addressed by singlecolor labeling and fluorescent double labeling using retrograde rAAVs injected into the bed nucleus of the stria terminalis(BST), anterior cortical amygdalar area(ACo), medial amygdaloid nucleus(MeA), and posteromedial cortical amygdaloid area(PMCo) in mice. We demonstrated the effectiveness of this AOB projection neuron labeling method and showed that the mitral cells of the AOB exhibited efferent projection dispersion characteristics similar to those of the MOB. Moreover, there were significant differences in the number of neurons projected to different brain regions, which indicated that each mitral cell in the AOB could project to a different number of neurons in different cortices. These results provide a circuitry basis to help understand the mechanism by which pheromone information is encoded and decoded in the AOS.

The Application of Deep Brain Stimulation for Parkinson’s Disease on the Motor Pathway:A Bibliometric Analysis across 10 Years

Background and Objective Since its initial report by James Parkinson in 1817,Parkinson’s disease(PD)has remained a central subject of research and clinical advancement.The disease is estimated to affect approximately 1%of adults aged 60 and above.Deep brain stimulation,emerging as an alternative therapy for end-stage cases,has offered a lifeline to numerous patients.This review aimed to analyze publications pertaining to the impact of deep brain stimulation on the motor pathway in patients with PD over the last decade.Methods Data were obtained from the Web of Science Core Collection through the library of Huazhong University of Science and Technology(China).The search strategy encompassed the following keywords:“deep brain stimulation”,“Parkinson’s disease”,“motor pathway”,and“human”,from January 1,2012,to December 1,2022.Additionally,this review visualized the findings using the Citespace software.Results The results indicated that the United States,the United Kingdom,Germany,and China were the primary contributors to this research field.University College London,Capital Medical University,and Maastricht University were the top 3 research institutions in the research area.Tom Foltynie ranked first with 6 publications,and the journals of Brain and Brain Stimulation published the greatest number of relevant articles.The prevailing research focal points in this domain,as determined by keywords“burst analysis”,“encompassed neuronal activity”,“nucleus”,“hyper direct pathway”,etc.Conclusion This study has provided a new perspective through bibliometric analysis of the deep brain stimulation therapy for treating patients with PD,which can shed light on future research to advance our comprehension of this particular field of study.

Rabies Virus Pseudotyped with CVS-N2C Glycoprotein as a Powerful Tool for Retrograde Neuronal Network Tracing

Efficient viral vectors for mapping and manipulating long-projection neuronal circuits are crucial in structural and functional studies of the brain. The SAD strain rabies virus with the glycoprotein gene deleted pseudotyped with the N2 C glycoprotein(SAD-RV(DG)-N2 C(G)) shows strong neuro-tropism in cell culture, but its in vivo efficiency for retrograde gene transduction and neuro-tropism have not been systematically characterized.We compared these features in different mouse brain regions for SAD-RV-N2 C(G) and two other widely-used retrograde tracers, SAD-RV(DG)-B19(G) and r AAV2-retro. We found that SAD-RV(DG)-N2 C(G) enhanced the infection efficiency of long-projecting neurons by^10 times but with very similar neuro-tropism, compared with SAD-RV(DG)-B19(G). On the other hand, SAD-RV(DG)-N2 C(G) had an infection efficiency comparable with r AAV2-retro, but a more restricted diffusion range, and broader tropism to different types and regions of longprojecting neuronal populations. These results demonstrate that SAD-RV(DG)-N2 C(G) can serve as an effective retrograde vector for studying neuronal circuits.

Preoperative Acute Sleep Deprivation Causes Postoperative Pain Hypersensitivity and Abnormal Cerebral Function

Preoperative sleep loss can amplify post-operative mechanical hyperalgesia.However,the underlying mechanisms are still largely unknown.In the current study,rats were randomly allocated to a control group and an acute sleep deprivation(ASD)group which experienced 6 h ASD before surgery.Then the variations in cerebral function and activity were investigated with multi-modal techniques,such as nuclear magnetic resonance,functional magnetic resonance imaging,c-Fos immunofluorescence,and electrophysiology.The results indicated that ASD induced hyperalgesia,and the metabolic kinetics were remarkably decreased in the striatum and midbrain.The functional connectivity(FC)between the nucleus accumbens(NAc,a subregion of the ventral striatum)and the ventrolateral periaqueductal gray(vLPAG)was significantly reduced,and the c-Fos expression in the NAc and the vLPAG was suppressed.Furthermore,the electrophysiological recordings demonstrated that both the neuronal activity in the NAc and the vLPAG,and the coherence of the NAc-vLPAG were suppressed in both resting and task states.This study showed that neuronal activity in the NAc and the vLPAG were weakened and the FC between the NAc and the vLPAG was also suppressed in rats with ASD-induced hyperalgesia.This study highlights the importance of preoperative sleep management for surgical patients.

Regional Metabolic Patterns of Abnormal Postoperative Behavioral Performance in Aged Mice Assessed by XH-NMR Dynamic Mapping Method

Abnormal postoperative neurobehavioral performance(APNP)is a common phenomenon in the early postoperative period.The disturbed homeostatic status of metabolites in the brain after anesthesia and surgery might make a significant contribution to APNP.The dynamic changes of metabolites in different brain regions after anesthesia and surgery,as well as their potential association with APNP are still not well understood.Here,we used a battery of behavioral tests to assess the effects of laparotomy under isoflurane anesthesia in aged mice,and investigated the metabolites in 12 different sub-regions of the brain at different time points using proton nuclear magnetic resonance('H-NMR)spectroscopy.The abnormal neurobehavioral performance occurred at 6 h and/or 9 h,and recovered at 24 h after anesthesia/surgery.Compared with the control group,the altered metabolite of the model group at 6 h was aspartate(Asp),and the difference was mainly displayed in the cortex;while significant changes at 9 h occurred predominantly in the cortex and hippocampus,and the corresponding metabolites were Asp and glutamate(Glu).All changes returned to baseline at 24 h.The altered metabolic changes could have occurred as a result of the acute APNP,and the metabolites Asp and Glu in the cortex and hippocampus could provide preliminary evidence for understanding the APNP process.

In-situ and one-step preparation of protein film in capillary column for open tubular capillary electrochromatography enantioseparation

In this wo rk,the phase-transitioned BSA(PTB)film using the mild and fast fabrication process adhered to the capillary inner wall uniformly,and the fabricated PTB film-coated capillary column was applied to realize open tubular capillary electrochromatography(OT-CEC)enantioseparation.The enantioseparation ability of PTB film-coated capillary was evaluated with eight pairs of chiral analytes including drugs and neurotransmitters,all achieving good resolution and symmetrical peak shape.For three consecutive runs,the relative standard deviations(RSD)of migration time for intra-day,inter-day,and column-tocolumn repeatability were in the range of 0.3%-3.5%,0.2%-4.9%and 2.1%-7.7%,respectively.Moreover,the PTB film-coated capillary column ran continuously over 300 times with high separation efficiency.Therefore,the coating method based on BSA self-assembly supramolecular film can be extended to the preparation of other proteinaceous capillary columns.