HIV-associated neurocognitive disorder (HAND), characterized by a wide spectrum of behavioral, cognitive, and motor dysfunctions, continues to affect approximately 50 % of HIV(+) patients despite the success of combination antiretroviral drug therapy (cART) in the periphery. Of note, potential toxicity of antiretroviral drugs in the central nervous system (CNS) remains remarkably underexplored and may contribute to the persistence of HAND in the cART era. Previous studies have shown antiretrovirals (ARVs) to be neurotoxic in the peripheral nervous system in vivo and in peripheral neurons in vitro. Alterations in lipid and protein metabolism, mitochondrial damage, and oxidative stress all play a role in peripheral ARV neurotoxicity. We hypothesized that ARVs also induce cellular stresses in the CNS, ultimately leading to neuronal damage and contributing to the changing clinical and pathological picture seen in HIV-positive patients in the cART era. In this report, we show that ARVs are neurotoxic in the CNS in both pigtail macaques and rats in vivo. Furthermore, in vitro, ARVs lead to accumulation of reactive oxygen species (ROS), and ultimately induction of neuronal damage and death. Whereas ARVs alone caused some activation of the endogenous antioxidant response in vitro, augmentation of this response by a fumaric acid ester, monomethyl fumarate (MMF), blocked ARV-induced ROS generation, and neuronal damage/death. These findings implicate oxidative stress as a contributor to the underlying mechanisms of ARV-induced neurotoxicity and will provide an access point for adjunctive therapies to complement ARV therapy and reduce neurotoxicity in this patient population.
Xenopus tropicalis has been developed as a model organism for developmental biology, providing a system offering both modern genetics and classical embryology. Recently, the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated (CRISPR/Cas) system for genome modification has provided an additional tool for Xenopus researchers to achieve simple and efficient targeted mutagenesis. Here, we provide insights into experimental design and procedures permitting successful application of this technique to Xenopus researchers, and offer a general strategy for performing loss-of-function assays in F0 and subsequently F1 embryos.
The cervical facet joint is implicated as one of the most common sources of chronic neck pain, owing to its rich nociceptive innervation and susceptibility to injurious mechanical loading. Injuries to the facet joint and its ligament can induce inflammation in the joint and spinal cord. Inflammatory molecules which are known to have a role in pain can also stimulate the integrated stress response (ISR). Therefore, we hypothesize that ISR is activated by facet joint injury in a rodent model of pain. To address this hypothesis, we assessed the expression of binding protein (BiP) (also known as growth-related protein 78 (GRP78)), a marker of endoplasmic reticulum stress response, in the dorsal root ganglion (DRG) after painful facet joint injury. In a rodent model of facet joint injury, dynamic distraction of the C6/C7 joint (injury, n = 12) was imposed; sham procedures were performed separately (sham, n = 8). Forepaw mechanical allodynia was assessed postoperatively for 7 days as a quantitative measure of pain symptoms. The C6 DRG was harvested and assessed for BiP expression using triple label immunofluorescent confocal microscopy and immunoblot analyses. BiP was significantly higher (p < 0.001) in the DRG after injury than sham and was expressed predominantly in neurons. Similarly, quantification of BiP by immunoblot demonstrated a significant 2.1-fold increase (p = 0.03) in injury compared to sham at day 7. Findings suggest neuronal stress activation is associated with painful facet joint injury, and that joint loading may directly mediate the behavior of DRG neurons in this class of injury. Inflammation contributes to persistent pain [5,34]. Cytokines and neurotrophic factors are upregulated in dorsal root ganglion (DRG) neurons in models of facet joint inflammation [20,21]. Lumbar neuropathic pain models exhibit glial cell activation, macrophage reactivity, and cytokine upregulation in the DRG and spinal cord [8,[32][33][34]. Glial activation can alter neuronal signaling and can also induce secretion of several pain mediators, including glutamate, pro-inflammatory cytokines and chemokines [19]. The release of glutamate and cytokines in the inflammatory cascade can also cause neuronal damage [2], and may trigger the integrated stress response (ISR). The integrated stress response is also known as the endoplasmic reticulum (ER) stress response or the unfolded protein response and is a protective cellular response that establishes protein homeostasis in response to a variety of injurious stimuli by increasing transcription and translation of gene products that regulate protein folding, degradation, delivery, and oxidation [16,18]. The ISR binding protein (BiP) is an ERresident chaperone that acts as a major regulator of the stress response [9,26]. While the mechanisms of activation of the ISR and the specific pathways of the neuronal stress response are receiving attention, their potential role in painful injury remains unexplored. KeywordsFacet joint distraction in the rat produces sustained spinal astr...
SUMMARY Mutations in the Pax6 gene cause ocular defects in both vertebrate and invertebrate animal species, and the disease aniridia in humans. Despite extensive experimentation on this gene in multiple species, including humans, we still do not understand the earliest effects on development mediated by this gene. This prompted us to develop pax6 mutant lines in Xenopus tropicalis taking advantage of the utility of the Xenopus system for examining early development and in addition to establish a model for studying the human disease aniridia in an accessible lower vertebrate. We have generated mutants in pax6 by using Transcription Activator-Like Effector Nuclease (TALEN) constructs for gene editing in X. tropicalis. Embryos with putative null mutations show severe eye abnormalities and changes in brain development, as assessed by changes in morphology and gene expression. One gene that we found is downregulated very early in development in these pax6 mutants is myc, a gene involved in pluripotency and progenitor cell maintenance and likely a mediator of some key pax6 functions in the embryo. Changes in gene expression in the developing brain and pancreas reflect other important functions of pax6 during development. In mutations with partial loss of pax6 function eye development is initially relatively normal but froglets show an underdeveloped iris, similar to the classic phenotype (aniridia) seen in human patients with PAX6 mutations. Other eye abnormalities observed in these froglets, including cataracts and corneal defects, are also common in human aniridia. The frog model thus allows us to examine the earliest deficits in eye formation as a result of pax6 lesions, and provides a useful model for understanding the developmental basis for the aniridia phenotype seen in humans.
Chronic neck pain from whiplash affects over 10 million people annually in the United States [1]. Many studies have indentified the facet joint as a possible source of neck pain due to its innervation by nociceptive fibers [2]. In particular, in vivo studies have reported that distraction of the facet joint and its capsule produces both persistent firing of capsule pain fibers and persistent behavioral hypersensitivity and pain symptoms (i.e. mechanical allodynia) [2,3].
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