Thermal escape box: A cost-benefit evaluation paradigm for investigating thermosensation and thermal pain.

Thermosensation, the ability to detect and estimate temperature, is an evolutionarily conserved process that is essential for survival. Thermosensing is impaired in various pain syndromes, resulting in thermal allodynia, the perception of an innocuous temperature as painful, or thermal hyperalgesia, an exacerbated perception of a painful thermal stimulus. Several behavioral assays exist to study thermosensation and thermal pain in rodents, however, most rely on reflexive withdrawal responses or the subjective quantification of spontaneous nocifensive behaviors. Here, we created a new apparatus, the thermal escape box, which can be attached to temperature-controlled plates and used to assess temperature-dependent effort-based decision-making. The apparatus consists of a light chamber with an opening that fits around temperature-controlled plates, and a small entryway into a dark chamber. A mouse must choose to stay in a brightly lit aversive area or traverse the plates to escape to the enclosed dark chamber. We quantified escape latencies of adult C57Bl/6 mice at different plate temperatures from video recordings and found they were significantly longer at 5 °C, 18 °C, and 52 °C, compared to 30 °C, a mouse's preferred ambient temperature. Differences in escape latencies were abolished in male Trpm8-/- mice and in male Trpv1-/- animals. Finally, we show that chronic constriction injury procedures or oxaliplatin treatement significantly increased escape latencies at cold temperatures compared to controls, the later of which was prevented by the analgesic meloxicam. This demonstrates the utility of this assay in detecting cold pain. Collectively, our study has identified a new and effective tool that uses cost-benefit valuations to study thermosensation and thermal pain.

Open-Label Sulforaphane Trial in FMR1 Premutation Carriers with Fragile-X-Associated Tremor and Ataxia Syndrome (FXTAS).

Fragile X (FMR1) premutation is a common mutation that affects about 1 in 200 females and 1 in 450 males and can lead to the development of fragile-X-associated tremor/ataxia syndrome (FXTAS). Although there is no targeted, proven treatment for FXTAS, research suggests that sulforaphane, an antioxidant present in cruciferous vegetables, can enhance mitochondrial function and maintain redox balance in the dermal fibroblasts of individuals with FXTAS, potentially leading to improved cognitive function. In a 24-week open-label trial involving 15 adults aged 60-88 with FXTAS, 11 participants successfully completed the study, demonstrating the safety and tolerability of sulforaphane. Clinical outcomes and biomarkers were measured to elucidate the effects of sulforaphane. While there were nominal improvements in multiple clinical measures, they were not significantly different after correction for multiple comparisons. PBMC energetic measures showed that the level of citrate synthase was higher after sulforaphane treatment, resulting in lower ATP production. The ratio of complex I to complex II showed positive correlations with the MoCA and BDS scores. Several mitochondrial biomarkers showed increased activity and quantity and were correlated with clinical improvements.

Strain and sex differences in somatosensation and sociability during experimental autoimmune encephalomyelitis.

Multiple Sclerosis (MS) is an immune-mediated disease that results in major locomotor deficits. However, recent studies have revealed that fatigue, slow processing speed, and memory impairment are the top variables impacting employment status for MS patients. These suggest that cognitive effects may have a greater impact on productivity, lifestyle, and quality of life than do disease-related motor deficits. However, these debilitating non-locomotive effects have been largely overlooked in rodent models of the disease, such as experimental autoimmune encephalomyelitis (EAE). We hypothesized that murine EAE can also be used to assess non-locomotive dysfunctions (mood, sociability, muscle strength, and balance), as well as potential biases in these dysfunctions due to sex and/or strain. We actively immunized male and female C57BL/6 (B6) and SJL mice for EAE and evaluated their performance on the Deacon's weight grip test, Kondziela's inverted screen test, Hall's rope grip test, manual von Frey test for somatic nociception, and a three-chamber social preference paradigm. We hypothesized that EAE progression is associated with changes in muscle strength, balance, pain, and sociability and that these variations are linked to sex and/or strain. Our results indicate that strain but not sex influenced differences in muscle strength and balance during EAE, and both sex and strain have an impact on mechanical nociception, regardless of EAE disease status. Furthermore, both sex and strain had complex effects on differences in sociability. In conclusion, testing these additional modalities during EAE helps to unveil other signs and symptoms that could be used to determine the efficacy of a drug or treatment in the modulation of a MS-like behavior.

Expression of IL-20 Receptor Subunit ß Is Linked to EAE Neuropathology and CNS Neuroinflammation.

Considerable clinical evidence supports that increased blood-brain barrier (BBB) permeability is linked to immune extravasation of CNS parenchyma during neuroinflammation. Although BBB permeability and immune extravasation are known to be provoked by vascular endothelial growth factor-A (i.e., VEGF-A) and C-X-C motif chemokine ligand 12 (CXCL12), respectively, the mechanisms that link both processes are still elusive. The interleukin-20 (i.e., IL-20) cytokine signaling pathway was previously implicated in VEGF-mediated angiogenesis and is known to induce cellular response by way of signaling through IL-20 receptor subunit ß (i.e., IL-20RB). Dysregulated IL-20 signaling is implicated in many inflammatory pathologies, but it's contribution to neuroinflammation has yet to be reported. We hypothesize that the IL-20 cytokine, and the IL cytokine subfamily more broadly, play a key role in CNS neuroinflammation by signaling through IL-20RB, induce VEGF activity, and enhance both BBB-permeability and CXCL12-mediated immune extravasation. To address this hypothesis, we actively immunized IL-20RB-/- mice and wild-type mice to induce experimental autoimmune encephalomyelitis (EAE) and found that IL-20RB-/- mice showed amelioration of disease progression compared to wild-type mice. Similarly, we passively immunized IL-20RB-/- mice and wild-type mice with myelin-reactive Th1 cells from either IL-20RB-/- and wild-type genotype. Host IL-20RB-/- mice showed lesser disease progression than wild-type mice, regardless of the myelin-reactive Th1 cells genotype. Using multianalyte bead-based immunoassay and ELISA, we found distinctive changes in levels of pro-inflammatory cytokines between IL-20RB-/- mice and wild-type mice at peak of EAE. We also found detectable levels of all cytokines of the IL-20 subfamily within CNS tissues and specific alteration to IL-20 subfamily cytokines IL-19, IL-20, and IL-24, expression levels. Immunolabeling of CNS region-specific microvessels confirmed IL-20RB protein at the spinal cord microvasculature and upregulation during EAE. Microvessels isolated from macaques CNS tissues also expressed IL-20RB. Moreover, we identified the expression of all IL-20 receptor subunits: IL-22 receptor subunit α-1 (IL-22RA1), IL-20RB, and IL-20 receptor subunit α (IL-20RA) in human CNS microvessels. Notably, human cerebral microvasculature endothelial cells (HCMEC/D3) treated with IL-1ß showed augmented expression of the IL-20 receptor. Lastly, IL-20-treated HCMEC/D3 showed alterations on CXCL12 apicobasal polarity consistent with a neuroinflammatory status. This evidence suggests that IL-20 subfamily cytokines may signal at the BBB via IL-20RB, triggering neuroinflammation.

Reliable Isolation of Central Nervous System Microvessels Across Five Vertebrate Groups.

Isolation of microvessels from the central nervous system (CNS) is commonly performed by combining cortical tissue from multiple animals, most often rodents. This approach limits the interrogation of blood-brain barrier (BBB) properties to the cortex and does not allow for individual comparison. This project focuses on the development of an isolation method that allows for the comparison of the neurovascular unit (NVU) from multiple CNS regions: cortex, cerebellum, optic lobe, hypothalamus, pituitary, brainstem, and spinal cord. Moreover, this protocol, originally developed for murine samples, was successfully adapted for use on CNS tissues from small and large vertebrate species from which we are also able to isolate microvessels from brain hemisphere white matter. This method, when paired with immunolabeling, allows for quantitation of protein expression and statistical comparison between individuals, tissue type, or treatment. We proved this applicability by evaluating changes in protein expression during experimental autoimmune encephalomyelitis (EAE), a murine model of a neuroinflammatory disease, multiple sclerosis. Additionally, microvessels isolated by this method could be used for downstream applications like qPCR, RNA-seq, and Western blot, among others. Even though this is not the first attempt to isolate CNS microvessels without the use of ultracentrifugation or enzymatic dissociation, it is unique in its adeptness for the comparison of single individuals and multiple CNS regions. Therefore, it allows for investigation of a range of differences that may otherwise remain obscure: CNS portions (cortex, cerebellum, optic lobe, brainstem, hypothalamus, pituitary, and spinal cord), CNS tissue type (gray or white matter), individuals, experimental treatment groups, and species.

Straightforward method for singularized and region-specific CNS microvessels isolation.

BACKGROUND: Current methods for murine brain microvasculature isolation requires the pooling of brain cortices while disregarding the rest of the CNS, making the analysis of single individuals non feasible. NEW METHOD: Efficient isolation of brain microvessels requires the elimination of meninges, vessels of high caliber vessels and choroid plexus, commonly done by rolling the over filter paper, but can't be done on other CNS regions. We overcome this hurdle by using a double-pronged pick, as well as elution and filtration through cell strainers after centrifugation. RESULTS: We were able to develop a region-specific murine CNS microvessels isolation, that allows for the comparison of the neurovascular unit from these regions both within the same individual and between multiple individuals and/or treatment groups without pooling. Additionally, we were able to adapt this method to macaque CNS tissue. COMPARISON WITH EXISTING METHOD(S): Although similar to a previously published method that requires no enzymatic dissociation and no ultracentrifugation, it does differ in its ability to isolate from a single experimental animal and from non-cortical tissues. However, it relies heavily on the researcher dissecting skills and careful elution and filtration of re-suspended samples. CONCLUSIONS: CNS region-specific microvessels comparison can inform of molecular and/or cellular differences that would otherwise be obscured by excluding non-cortical tissue. Additionally, it allows for the unmasking of variations between individuals that remained hidden when pooling of multiple samples is the norm. Lastly, isolation of region-specific microvessels for non-human primate CNS allows for more translationally relevant studies of the BBB.