Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Inquimbert, Perrine; Moll, Martin; Kohno, Tatsuro; Scholz, Joachim

doi:10.3791/50313-v

Cited by 3 publications

(3 citation statements)

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“…Adeno‐associated virus (AAV) containing a gene‐encoding hM4Di under the control of the CaMKIIα promoter (pAAV‐CaMKIIα‐hM4D(Gi)‐EGFP or pAAV‐CaMKIIα‐hM4D(Gi)‐mCherry) were purchased from HanBio (Shanghai, China). The viral vector microinjection procedure followed the methods described in previous studies 24,25 . Mice were anesthetized with isoflurane inhalation in 100% oxygen and positioned prone on the operating table.…”

Section: Methodsmentioning

confidence: 99%

“…The viral vector microinjection procedure followed the methods described in previous studies. 24,25 Mice were anesthetized with isoflurane inhalation in 100% oxygen and positioned prone on the operating table. Following a midline incision at the base of the rib cage, the spinal cord was exposed by separating the overlying fascia and removing the dorsal part of the vertebra using laminectomy forceps.…”

Section: Intraspinal Cord Microinjectionmentioning

confidence: 99%

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SIRT1 mediates the excitability of spinal CaMKIIα‐positive neurons and participates in neuropathic pain by controlling Nav1.3

Wang,

Zhang,

et al. 2024

CNS Neurosci Ther

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AimsNeuropathic pain is a common chronic pain disorder, which is largely attributed to spinal central sensitization. Calcium/calmodulin‐dependent protein kinase II alpha (CaMKIIα) activation in the spinal dorsal horn (SDH) is a major contributor to spinal sensitization. However, the exact way that CaMKIIα‐positive (CaMKIIα+) neurons in the SDH induce neuropathic pain is still unclear. This study aimed to explore the role of spinal CaMKIIα+ neurons in neuropathic pain caused by chronic constriction injury (CCI) and investigate the potential epigenetic mechanisms involved in CaMKIIα+ neuron activation.MethodsCCI‐induced neuropathic pain mice model, Sirt1loxP/loxP mice, and chemogenetic virus were used to investigate whether the activation of spinal CaMKIIα+ neurons is involved in neuropathic pain and its involved mechanism. Transcriptome sequence, western blotting, qRT‐PCR, and immunofluorescence analysis were performed to assay the expression of related molecules and activation of neurons. Co‐immunoprecipitation was used to observe the binding relationship of protein. Chromatin immunoprecipitation (ChIP)‐PCR was applied to analyze the acetylation of histone H3 in the Scn3a promoter region.ResultsThe expression of sodium channel Nav1.3 was increased and the expression of SIRT1 was decreased in the spinal CaMKIIα+ neurons of CCI mice. CaMKIIα neurons became overactive after CCI, and inhibiting their activation relieved CCI‐induced pain. Overexpression of SIRT1 reversed the increase of Nav1.3 and alleviated pain, while knockdown of SIRT1 or overexpression of Nav1.3 promoted CaMKIIα+ neuron activation and induced pain. By knocking down spinal SIRT1, the acetylation of histone H3 in the Scn3a (encoding Nav1.3) promoter region was increased, leading to an increased expression of Nav1.3.ConclusionThe findings suggest that an aberrant reduction of spinal SIRT1 after nerve injury epigenetically increases Nav1.3, subsequently activating CaMKIIα+ neurons and causing neuropathic pain.

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Section: Methodsmentioning

confidence: 99%

Section: Intraspinal Cord Microinjectionmentioning

confidence: 99%

SIRT1 mediates the excitability of spinal CaMKIIα‐positive neurons and participates in neuropathic pain by controlling Nav1.3

Wang,

Zhang,

et al. 2024

CNS Neurosci Ther

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“…Injection of lentivirus in the lower thoracic spinal cord dorsal horn was performed as described in previous studies. 14,15 Briefly, using sodium pentobarbital (40 mg/kg sodium pentobarbital) to induce deep anesthesia, skin incisions corresponding to the thoracic T8-10 spinal cord segment were performed to expose vertebrae under semisterile conditions. Carefully drill two 2 mm diameter holes on two sides of the vertebral plate of thoracic T9.…”

Section: Stereotaxic Injection Of Virus In the Lower Thoracic Spinal ...mentioning

confidence: 99%

LncTCONS_00058568 is involved in the pathophysiologic processes mediated by P2X7R in the lower thoracic spinal cord after acute kidney injury

Wang,

Chen,

Liu

et al. 2024

The FASEB Journal

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Acute kidney injury (AKI), a prevalent clinical syndrome, involves the participation of the nervous system in neuroimmune regulation. However, the intricate molecular mechanism that governs renal function regulation by the central nervous system (CNS) is complex and remains incompletely understood. In the present study, we found that the upregulated expression of lncTCONS_00058568 in lower thoracic spinal cord significantly ameliorated AKI‐induced renal tissue injury, kidney morphology, inflammation and apoptosis, and suppressed renal sympathetic nerve activity. Mechanistically, the purinergic ionotropic P2X7 receptor (P2X7R) was overexpressed in AKI rats, whereas lncTCONS_00058568 was able to suppress the upregulation of P2X7R. In addition, RNA sequencing data revealed differentially expressed genes associated with nervous system inflammatory responses after lncTCONS_00058568 was overexpressed in AKI rats. Finally, the overexpression of lncTCONS_00058568 inhibited the activation of PI3K/Akt and NF‐κB signaling pathways in spinal cord. Taken together, the results from the present study show that lncTCONS_00058568 overexpression prevented renal injury probably by inhibiting sympathetic nerve activity mediated by P2X7R in the lower spinal cord subsequent to I/R‐AKI.

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Development and characterization of a Gucy2d-cre mouse to selectively manipulate a subset of inhibitory spinal dorsal horn interneurons

Serafin,

Yoo,

et al. 2024

PLoS ONE

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Recent transcriptomic studies identified Gucy2d (encoding guanylate cyclase D) as a highly enriched gene within inhibitory dynorphin interneurons in the mouse spinal dorsal horn. To facilitate investigations into the role of the Gucy2d+ population in somatosensation, Gucy2d-cre transgenic mice were created to permit chemogenetic or optogenetic manipulation of this subset of spinal neurons. Gucy2d-cre mice created via CRISPR/Cas9 genomic knock-in were bred to mice expressing a cre-dependent reporter (either tdTomato or Sun1.GFP fusion protein), and the resulting offspring were characterized. Surprisingly, a much wider population of spinal neurons was labeled by cre-dependent reporter expression than previous mRNA-based studies would suggest. Although the cre-dependent reporter expression faithfully labeled ~75% of cells expressing Gucy2d mRNA in the adult dorsal horn, it also labeled a substantial number of additional inhibitory neurons in which no Gucy2d or Pdyn mRNA was detected. Moreover, cre-dependent reporter was also expressed in various regions of the brain, including the spinal trigeminal nucleus, cerebellum, thalamus, somatosensory cortex, and anterior cingulate cortex. Injection of AAV-CAG-FLEX-tdTomato viral vector into adult Gucy2d-cre mice produced a similar pattern of cre-dependent reporter expression in the spinal cord and brain, which excludes the possibility that the unexpected reporter-labeling of cells in the deep dorsal horn and brain was due to transient Gucy2d expression during early stages of development. Collectively, these results suggest that Gucy2d is expressed in a wider population of cells than previously thought, albeit at levels low enough to avoid detection with commonly used mRNA-based assays. Therefore, it is unlikely that these Gucy2d-cre mice will permit selective manipulation of inhibitory signaling mediated by spinal dynorphin interneurons, but this novel cre driver line may nevertheless be useful to target a broader population of inhibitory spinal dorsal horn neurons.

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Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Cited by 3 publications

References 9 publications

SIRT1 mediates the excitability of spinal CaMKIIα‐positive neurons and participates in neuropathic pain by controlling Nav1.3

SIRT1 mediates the excitability of spinal CaMKIIα‐positive neurons and participates in neuropathic pain by controlling Nav1.3

LncTCONS_00058568 is involved in the pathophysiologic processes mediated by P2X7R in the lower thoracic spinal cord after acute kidney injury

Development and characterization of a Gucy2d-cre mouse to selectively manipulate a subset of inhibitory spinal dorsal horn interneurons

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