Techniques for Intracranial Stereotaxic Injections of Adeno‐Associated Viral Vectors in Adult Mice

Keiser, Megan S.; Davidson, Beverly L.

doi:10.1002/cpmo.57

Cited by 15 publications

(10 citation statements)

References 8 publications

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“…For intracortical injections, a borehole was created on the left side using a high-speed drill at coordinates relative to the bregma (X = 1.0 mm, Y = 0.80 mm). The syringe was connected to a microinjection pump, the needle was inserted into the brain through the burr hole (Z = 1.5 mm from the bone surface), and 1 µl of Gal-3 knockdown adeno-associated virus (AAV) (Hanheng China) was infused [ 46 ]. The Gal-3 and control shRNA sequences are shown in Table 2 .…”

Section: Methodsmentioning

confidence: 99%

D-allose Inhibits TLR4/PI3K/AKT Signaling to Attenuate Neuroinflammation and Neuronal Apoptosis by Inhibiting Gal-3 Following Ischemic Stroke

Luo,

Cheng,

et al. 2023

Biol Proced Online

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Background Ischemic stroke (IS) occurs when a blood vessel supplying the brain becomes obstructed, resulting in cerebral ischemia. This type of stroke accounts for approximately 87% of all strokes. Globally, IS leads to high mortality and poor prognosis and is associated with neuroinflammation and neuronal apoptosis. D-allose is a bio-substrate of glucose that is widely expressed in many plants. Our previous study showed that D-allose exerted neuroprotective effects against acute cerebral ischemic/reperfusion (I/R) injury by reducing neuroinflammation. Here, we aimed to clarify the beneficial effects D-allose in suppressing IS-induced neuroinflammation damage, cytotoxicity, neuronal apoptosis and neurological deficits and the underlying mechanism in vitro and in vivo. Methods In vivo, an I/R model was induced by middle cerebral artery occlusion and reperfusion (MCAO/R) in C57BL/6 N mice, and D-allose was given by intraperitoneal injection within 5 min after reperfusion. In vitro, mouse hippocampal neuronal cells (HT-22) with oxygen–glucose deprivation and reperfusion (OGD/R) were established as a cell model of IS. Neurological scores, some cytokines, cytotoxicity and apoptosis in the brain and cell lines were measured. Moreover, Gal-3 short hairpin RNAs, lentiviruses and adeno-associated viruses were used to modulate Gal-3 expression in neurons in vitro and in vivo to reveal the molecular mechanism. Results D-allose alleviated cytotoxicity, including cell viability, LDH release and apoptosis, in HT-22 cells after OGD/R, which also alleviated brain injury, as indicated by lesion volume, brain edema, neuronal apoptosis, and neurological functional deficits, in a mouse model of I/R. Moreover, D-allose decreased the release of inflammatory factors, such as IL-1β, IL-6 and TNF-α. Furthermore, the expression of Gal-3 was increased by I/R in wild-type mice and HT-22 cells, and this factor further bound to TLR4, as confirmed by three-dimensional structure prediction and Co-IP. Silencing the Gal-3 gene with shRNAs decreased the activation of TLR4 signaling and alleviated IS-induced neuroinflammation, apoptosis and brain injury. Importantly, the loss of Gal-3 enhanced the D-allose-mediated protection against I/R-induced HT-22 cell injury, inflammatory insults and apoptosis, whereas activation of TLR4 by the selective agonist LPS increased the degree of neuronal injury and abolished the protective effects of D-allose. Conclusions In summary, D-allose plays a crucial role in inhibiting inflammation after IS by suppressing Gal-3/TLR4/PI3K/AKT signaling pathway in vitro and in vivo.

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Section: Methodsmentioning

confidence: 99%

D-allose Inhibits TLR4/PI3K/AKT Signaling to Attenuate Neuroinflammation and Neuronal Apoptosis by Inhibiting Gal-3 Following Ischemic Stroke

Luo,

Cheng,

et al. 2023

Biol Proced Online

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“…Five-week-old mice were prepared for stereotaxic injection. The detailed protocols have been described previously [ 24 ] with minor modifications. Briefly, animals were placed into the anesthesia induction chamber with 2.5% isoflurane and then immobilized on a stereotaxic apparatus.…”

Section: Methodsmentioning

confidence: 99%

Deficiency of ROS‐Activated TRPM2 Channel Protects Neurons from Cerebral Ischemia‐Reperfusion Injury through Upregulating Autophagy

Liu

et al. 2021

Oxidative Medicine and Cellular Longevity

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Cerebral ischemia-reperfusion (I-R) transiently increased autophagy by producing excessively reactive oxygen species (ROS); on the other hand, activated autophagy would remove ROS-damaged mitochondria and proteins, which led to cell survival. However, the regulation mechanism of autophagy activity during cerebral I-R is still unclear. In this study, we found that deficiency of the TRPM2 channel which is a ROS sensor significantly decreased I-R-induced neuronal damage. I-R transiently increased autophagy activity both in vitro and in vivo. More importantly, TRPM2 deficiency decreased I-R-induced neurological deficit score and infarct volume. Interestingly, our results indicated that TRPM2 deficiency could further activate AMPK rather than Beclin1 activity, suggesting that TRPM2 inhibits autophagy by regulating the AMPK/mTOR pathway in I-R. In conclusion, our study reveals that ROS-activated TRPM2 inhibits autophagy by downregulating the AMPK/mTOR pathway, which results in neuronal death induced by cerebral I-R, further supporting that TRPM2 might be a potential drug target for cerebral ischemic injury therapy.

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“…Mice were anesthetized intraperitoneally with chloral hydrate (4%, 0.01 ml/g) before the intracranial stereotaxic injection of AAV or NS through the mouse stereotaxic apparatus (RWD LIFE SCIENCE). Details of the stereotaxic injection were described previously (Keiser, Chen, & Davidson, 2018). Briefly, we made a longitudinal incision to expose the bregma, set this point as zero, and determined the hippocampus region from this point (AP ± 2 mm, ML ± 2 mm, DV ± 2 mm).…”

Section: Methodsmentioning

confidence: 99%

Effect of Sox10 on remyelination of the hippocampus in cuprizone‐induced demyelinated mice

Shao

Ding

et al. 2020

Brain and Behavior

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Techniques for Intracranial Stereotaxic Injections of Adeno‐Associated Viral Vectors in Adult Mice

Cited by 15 publications

References 8 publications

D-allose Inhibits TLR4/PI3K/AKT Signaling to Attenuate Neuroinflammation and Neuronal Apoptosis by Inhibiting Gal-3 Following Ischemic Stroke

D-allose Inhibits TLR4/PI3K/AKT Signaling to Attenuate Neuroinflammation and Neuronal Apoptosis by Inhibiting Gal-3 Following Ischemic Stroke

Deficiency of ROS‐Activated TRPM2 Channel Protects Neurons from Cerebral Ischemia‐Reperfusion Injury through Upregulating Autophagy

Effect of Sox10 on remyelination of the hippocampus in cuprizone‐induced demyelinated mice

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