Use of adeno-associated viruses for transgenic modulation of microglia structure and function: A review of technical considerations and challenges.

Microglia play a central role in the etiology of many neuropathologies. Transgenic tools are a powerful experiment approach to gain reliable and specific control over microglia function. Adeno-associated virus (AAVs) vectors are already an indispensable tool in neuroscience research. Despite ubiquitous use of AAVs and substantial interest in the role of microglia in the study of central nervous system (CNS) function and disease, transduction of microglia using AAVs is seldom reported. This review explores the challenges and advancements made in using AAVs for expressing transgenes in microglia. First, we will examine the functional anatomy of the AAV capsid, which will serve as a basis for subsequent discussions of studies exploring the relationship between capsid mutations and microglia transduction efficacy. After outlining the functional anatomy of AAVs, we will consider the experimental evidence demonstrating AAV-mediated transduction of microglia and microglia-like cell lines followed by an examination of the most promising experimental approaches identified in the literature. Finally, technical limitations will be considered in future applications of AAV experimental approaches.

Interleukin-1beta and inflammasome expression in spinal cord following chronic constriction injury in male and female rats.

Females represent a majority of chronic pain patients and show greater inflammatory immune responses in human chronic pain patient populations as well as in animal models of neuropathic pain. Recent discoveries in chronic pain research have revealed sex differences in inflammatory signaling, a key component of sensory pathology in chronic neuropathic pain, inviting more research into the nuances of these sex differences. Here we use the chronic constriction injury (CCI) model to explore similarities and differences in expression and production of Inflammatory cytokine IL-1beta in the lumbar spinal cord, as well as its role in chronic pain. We have discovered that intrathecal IL-1 receptor antagonist reverses established pain in both sexes, and increased gene expression of inflammasome NLRP3 is specific to microglia and astrocytes rather than neurons, while IL-1beta is specific to microglia in both sexes. We report several sex differences in the expression level of the genes coding for IL-1beta, as well as the four inflammasomes responsible for IL-1beta release: NLRP3, AIM2, NLRP1, and NLRC4 in the spinal cord. Total mRNA, but not protein expression of IL-1beta is greater in females than males after CCI. Also, while CCI increases all four inflammasomes in both sexes, there are sex differences in relative levels of inflammasome expression. NLRP3 and AIM2 are more highly expressed in females, whereas NLRP1 expression is greater in males.

Voluntary wheel running prevents formation of membrane attack complexes and myelin degradation after peripheral nerve injury.

Regular aerobic activity is associated with a reduced risk of chronic pain in humans and rodents. Our previous studies in rodents have shown that prior voluntary wheel running can normalize redox signaling at the site of peripheral nerve injury, attenuating subsequent neuropathic pain. However, the full extent of neuroprotection offered by voluntary wheel running after peripheral nerve injury is unknown. Here, we show that six weeks of voluntary wheel running prior to chronic constriction injury (CCI) reduced the terminal complement membrane attack complex (MAC) at the sciatic nerve injury site. This was associated with increased expression of the MAC inhibitor CD59. The levels of upstream complement components (C3) and their inhibitors (CD55, CR1 and CFH) were altered by CCI, but not increased by voluntary wheel running. Since MAC can degrade myelin, which in turn contributes to neuropathic pain, we evaluated myelin integrity at the sciatic nerve injury site. We found that the loss of myelinated fibers and decreased myelin protein which occurs in sedentary rats following CCI was not observed in rats with prior running. Substitution of prior voluntary wheel running with exogenous CD59 also attenuated mechanical allodynia and reduced MAC deposition at the nerve injury site, pointing to CD59 as a critical effector of the neuroprotective and antinociceptive actions of prior voluntary wheel running. This study links attenuation of neuropathic pain by prior voluntary wheel running with inhibition of MAC and preservation of myelin integrity at the sciatic nerve injury site.

Combining RNAscope and immunohistochemistry to visualize inflammatory gene products in neurons and microglia.

A challenge for central nervous system (CNS) tissue analysis in neuroscience research has been the difficulty to codetect and colocalize gene and protein expression in the same tissue. Given the importance of identifying gene expression relative to proteins of interest, for example, cell-type specific markers, we aimed to develop a protocol to optimize their codetection. RNAscope fluorescent in situ hybridization (FISH) combined with immunohistochemistry (IHC) in fixed (CNS) tissue sections allows for reliable quantification of gene transcripts of interest within IHC-labeled cells. This paper describes a new method for simultaneous visualization of FISH and IHC in thicker (14-µm), fixed tissue samples, using spinal cord sections. This method's effectiveness is shown by the cell-type-specific quantification of two genes, namely the proinflammatory cytokine interleukin-1beta (IL-1b) and the inflammasome NLR family pyrin domain containing 3 (NLRP3). These genes are challenging to measure accurately using immunohistochemistry (IHC) due to the nonspecificity of available antibodies and the hard-to-distinguish, dot-like visualizations of the labeled proteins within the tissue. These measurements were carried out in spinal cord sections after unilateral chronic constriction injury of the sciatic nerve to induce neuroinflammation in the spinal cord. RNAscope is used to label transcripts of genes of interest and IHC is used to label cell-type specific antigens (IBA1 for microglia, NeuN for neurons). This combination allowed for labeled RNA transcripts to be quantified within cell-type specific boundaries using confocal microscopy and standard image analysis methods. This method makes it easy to answer empirical questions that are intractable with standard IHC or in situ hybridization alone. The method, which has been optimized for spinal cord tissue and to minimize tissue preparation time and costs, is described in detail from tissue collection to image analysis. Further, the relative expression changes in inflammatory genes NLRP3 and IL-1b in spinal cord microglia vs. neurons of somatotopically relevant laminae are described for the first time.

Mechanisms of microglia-mediated synapse turnover and synaptogenesis.

Microglia shape synaptic connections among neurons of the central nervous system (CNS) during development and adulthood. In this review, the actions by which they facilitate pruning, refinement, and new synaptic development throughout the lifespan are considered, along with the molecular mechanisms by which neurons and microglia communicate to guide these actions. Microglia survey neuronal activity and selectively modify synaptic connections at the level of individual dendrites and synapses. This is important given that microglia are necessary for a healthy nervous system capable of learning and other neural phenomena based on synaptic modifications and can also cause pathological synaptic disfunctions in immunologically driven neurodegenerative diseases. Understanding how microglia directly shape synaptic connections between neurons yields a more complete understanding of normal neuroplasticity and provides new routes for understanding disease states.

Preconditioning by voluntary wheel running attenuates later neuropathic pain via nuclear factor E2-related factor 2 antioxidant signaling in rats.

ABSTRACT: Animal and human studies have shown that exercise prior to nerve injury prevents later chronic pain, but the mechanisms of such preconditioning remain elusive. Given that exercise acutely increases the formation of free radicals, triggering antioxidant compensation, we hypothesized that voluntary running preconditioning would attenuate neuropathic pain by supporting redox homeostasis after sciatic nerve injury in male and female rats. We show that 6 weeks of voluntary wheel running suppresses neuropathic pain development induced by chronic constriction injury across both sexes. This attenuation was associated with reduced nitrotyrosine immunoreactivity-a marker for peroxynitrite-at the sciatic nerve injury site. Our data suggest that prior voluntary wheel running does not reduce the production of peroxynitrite precursors, as expression levels of inducible nitric oxide synthase and NADPH oxidase 2 were unchanged. Instead, voluntary wheel running increased superoxide scavenging by elevating expression of superoxide dismutases 1 and 2. Prevention of neuropathic pain was further associated with the activation of the master transcriptional regulator of the antioxidant response, nuclear factor E2-related factor 2 (Nrf2). Six weeks of prior voluntary wheel running increased Nrf2 nuclear translocation at the sciatic nerve injury site; in contrast, 3 weeks of prior wheel running, which failed to prevent neuropathic pain, had no effect on Nrf2 nuclear translocation. The protective effects of prior voluntary wheel running were mediated by Nrf2, as suppression was abolished across both sexes when Nrf2 activation was blocked during the 6-week running phase. This study provides insight into the mechanisms by which physical activity may prevent neuropathic pain. Preconditioning by voluntary wheel running, terminated prior to nerve injury, suppresses later neuropathic pain in both sexes, and it is modulated through the activation of Nrf2-antioxidant signaling.

SARS-CoV-2 spike S1 subunit induces neuroinflammatory, microglial and behavioral sickness responses: Evidence of PAMP-like properties.

SARS-CoV-2 infection produces neuroinflammation as well as neurological, cognitive (i.e., brain fog), and neuropsychiatric symptoms (e.g., depression, anxiety), which can persist for an extended period (6 months) after resolution of the infection. The neuroimmune mechanism(s) that produces SARS-CoV-2-induced neuroinflammation has not been characterized. Proposed mechanisms include peripheral cytokine signaling to the brain and/or direct viral infection of the CNS. Here, we explore the novel hypothesis that a structural protein (S1) derived from SARS-CoV-2 functions as a pathogen-associated molecular pattern (PAMP) to induce neuroinflammatory processes independent of viral infection. Prior evidence suggests that the S1 subunit of the SARS-CoV-2 spike protein is inflammatory in vitro and signals through the pattern recognition receptor TLR4. Therefore, we examined whether the S1 subunit is sufficient to drive 1) a behavioral sickness response, 2) a neuroinflammatory response, 3) direct activation of microglia in vitro, and 4) activation of transgenic human TLR2 and TLR4 HEK293 cells. Adult male Sprague-Dawley rats were injected intra-cisterna magna (ICM) with vehicle or S1. In-cage behavioral monitoring (8 h post-ICM) demonstrated that S1 reduced several behaviors, including total activity, self-grooming, and wall-rearing. S1 also increased social avoidance in the juvenile social exploration test (24 h post-ICM). S1 increased and/or modulated neuroimmune gene expression (Iba1, Cd11b, MhcIIα, Cd200r1, Gfap, Tlr2, Tlr4, Nlrp3, Il1b, Hmgb1) and protein levels (IFNγ, IL-1ß, TNF, CXCL1, IL-2, IL-10), which varied across brain regions (hypothalamus, hippocampus, and frontal cortex) and time (24 h and 7d) post-S1 treatment. Direct exposure of microglia to S1 resulted in increased gene expression (Il1b, Il6, Tnf, Nlrp3) and protein levels (IL-1ß, IL-6, TNF, CXCL1, IL-10). S1 also activated TLR2 and TLR4 receptor signaling in HEK293 transgenic cells. Taken together, these findings suggest that structural proteins derived from SARS-CoV-2 might function independently as PAMPs to induce neuroinflammatory processes via pattern recognition receptor engagement.

Suppression of active phase voluntary wheel running in male rats by unilateral chronic constriction injury: Enduring therapeutic effects of a brief treatment of morphine combined with TLR4 or P2X7 antagonists.

The present series of studies examine the impact of systemically administered therapeutics on peripheral nerve injury (males; unilateral sciatic chronic constriction injury [CCI])-induced suppression of voluntary wheel running, across weeks after dosing cessation. Following CCI, active phase running distance and speed are suppressed throughout the 7-week observation period. A brief course of morphine, however, increased active phase running distance and speed throughout this same period, an effect apparent only in sham rats. For CCI rats, systemic co-administration of morphine with antagonists of either P2X7 (A438079) or TLR4 ((+)-naloxone) (receptors critical to the activation of NLRP3 inflammasomes and consequent inflammatory cascades) returned running behavior of CCI rats to that of shams through 5+ weeks after dosing ceased. This is a striking difference in effect compared to our prior CCI allodynia results using systemic morphine plus intrathecal delivery of these same antagonists, wherein a sustained albeit partial suppression of neuropathic pain was observed. This may point to actions of the systemic drugs at multiple sites along the neuraxis, modulating injury-induced, inflammasome-mediated effects at the injured sciatic nerve and/or dorsal root ganglia, spinal cord, and potentially higher levels. Given that our data to date point to morphine amplifying neuroinflammatory processes put into motion by nerve injury, it is intriguing to speculate that co-administration of TLR4 and/or P2X7 antagonists can intervene in these inflammatory processes in a beneficial way. That is, that systemic administration of such compounds may suppress inflammatory damage at multiple sites, rapidly and persistently returning neuropathic animals to sham levels of response.

Oxycodone, fentanyl, and morphine amplify established neuropathic pain in male rats.

Opioids are widely prescribed for chronic pain, including neuropathic pain, despite growing evidence of long-term harm. Previous preclinical studies have documented exacerbation of nociceptive hypersensitivity, including that induced by peripheral nerve injury, by morphine. The present series of behavioral studies sought to replicate and extend our prior research, which demonstrated a multimonth exacerbation of nociceptive hypersensitivity by a 5-day course of morphine initiated 10 days after nerve injury. The current studies demonstrate that enduring exacerbation of nociceptive hypersensitivity is not restricted to morphine, but rather is also created by the clinically relevant opioids fentanyl and oxycodone when these are likewise administered for 5 days beginning 10 days after nerve injury. Furthermore, enduring exacerbation of nociceptive hypersensitivity is also observed when the same dosing regimen for either morphine, fentanyl, or oxycodone begins 1 month after nerve injury. Finally, a striking result from these studies is that no such exacerbation of nociceptive hypersensitivity occurs when either morphine, fentanyl, or oxycodone dosing begins at the time of nerve injury. These results extend our previous findings that morphine exacerbates nociceptive hypersensitivity to the clinically relevant opioids fentanyl and oxycodone when administered after the development of nociceptive hypersensitivity, while also providing possible clinically relevant insight into when these opioids can be safely administered and not exacerbate neuropathic pain.