Hypersensitivity In Mice Research Articles

ZnO Nanoparticles as Potent Inducers of Dermal Immunosuppression in Contact Hypersensitivity in Mice.

Nanosized zinc oxide (nZnO) metal particles are used in skin creams and sunscreens to enhance their texture and optical properties as UV filters. Despite their common use, little is known about the molecular mechanisms of nZnO exposure on damaged skin. We studied the effects of topically applied nZnO particles on allergic skin inflammation in an oxazolone (OXA)-induced contact hypersensitivity (CHS) mouse model. We investigated whether exposure to nZnO during the sensitization or challenge phase would induce immunological changes and modulate transcriptional responses. We followed skin thickness, cellular infiltration, and changes in the local transcriptome up to 28 days after the challenge. The responses peaked at 24 h and were fully resolved by 28 days. Co-exposure to nZnO and hapten did not interfere with the formation of the sensitization process. Conversely, during the hapten challenge, the application of nZnO fully suppressed the development of the CHS response by the inhibition of pro-inflammatory pathways, secretion of pro-inflammatory cytokines, and proliferation of immune cells. In differentiated and stimulated THP-1 cells and the CHS mouse model, we found that nZnO particles and Zn ions contributed to anti-inflammatory responses. The immunosuppressive properties of nZnO in inflamed skin are mediated by impaired IL-1R-, CXCR2-, and LTB4-mediated pathways. nZnO-induced dermal immunosuppression may be beneficial for individuals with contact allergies who use nZnO-containing cosmetic products. Our findings also provide a deeper understanding of the mechanisms of nZnO, which could be considered when developing nanoparticle-containing skin products.

Suppression of Visceral Nociception by Selective C-Fiber Transmission Block Using Temporal Interference Sinusoidal Stimulation.

Chronic visceral pain management remains challenging due to limitations in selective targeting of C-fiber nociceptors. This study investigates temporal interference stimulation (TIS) on dorsal root ganglia (DRG) as a novel approach for selective C-fiber transmission block. We employed (1) GCaMP6 recordings in mouse whole DRG using a flexible, transparent microelectrode array for visualizing L6 DRG neuron activation, (2) ex vivo single-fiber recordings to assess sinusoidal stimulation effects on peripheral nerve axons, (3) in vivo behavioral assessment measuring visceromotor responses (VMR) to colorectal distension in mice, including a TNBS-induced visceral hypersensitivity model, and (4) immunohistological analysis to evaluate immediate immune responses in DRG following TIS. We demonstrated that TIS (2000 Hz and 2020 Hz carrier frequencies) enabled tunable activation of L6 DRG neurons, with the focal region adjustable by altering stimulation amplitude ratios. Low-frequency (20-50 Hz) sinusoidal stimulation effectively blocked C-fiber and Aδ-fiber transmission while sparing fast-conducting A-fibers, with 20 Hz showing highest efficacy. TIS of L6 DRG reversibly suppressed VMR to colorectal distension in both control and TNBS-induced visceral hypersensitive mice. The blocking effect was fine-tunable by adjusting interfering stimulus signal amplitude ratios. No apparent immediate immune responses were observed in DRG following hours-long TIS. In conclusion, TIS on lumbosacral DRG demonstrates promise as a selective, tunable approach for managing chronic visceral pain by effectively blocking C-fiber transmission. This technique addresses limitations of current neuromodulation methods and offers potential for more targeted relief in chronic visceral pain conditions.

Analgesic effect of a cholinergic agonist (carbachol) in a sural nerve ligation-induced hypersensitivity mouse model

ABSTRACT Objectives Neuropathic pain is characterized by long-lasting, intractable pain. Sciatic nerve ligation is often used as an animal model of neuropathic pain, and the spared nerve injury (SNI) model, in which the common peroneal nerve (CPN) and tibial nerve (TN) are ligated, is widely used. In the present study, we evaluated the analgesic effect of a cholinergic agonist, carbachol, on a neuropathic pain model prepared by sural nerve (SN) ligation in mice. Methods The SN was tightly ligated as a branch of the sciatic nerve. Mechanical and thermal allodynia, and hyperalgesia were assessed using von Frey filaments and heat from a hot plate. The analgesic effects of intracerebroventricularly-administered morphine and carbachol were compared. Results SN ligation resulted in a significant decrease in pain threshold for mechanical stimulation 1 day after ligation. In response to thermal stimulation, allodynia was observed at 50°C and hyperalgesia at 53 and 56°C 3 days after ligation. Content of thiobarbituric acid reactive substances (TBARS) in the spinal cord increased significantly at 6 and 12 h after ligation. Acetylcholine content of the spinal cord also increased at 5 and 7 days after ligation. Intracerebroventricular administration of carbachol at 7 days after ligation produced a marked analgesic effect against mechanical and thermal stimuli, which was stronger and longer-lasting than morphine at all experimental time points. Conclusion These findings suggest that cholinergic nerves are involved in allodynia and hyperalgesia of the SN ligation neuropathic pain model.

A CD25-biased interleukin-2 for autoimmune therapy engineered via a semi-synthetic organism

BackgroundNatural cytokines are poorly suited as therapeutics for systemic administration due to suboptimal pharmacological and pharmacokinetic (PK) properties. Recombinant human interleukin-2 (rhIL-2) has shown promise for treatment of autoimmune (AI) disorders yet exhibits short systemic half-life and opposing immune responses that negate an appropriate therapeutic index.MethodsA semi-synthetic microbial technology platform was used to engineer a site-specifically pegylated form of rhIL-2 with enhanced PK, specificity for induction of immune-suppressive regulatory CD4 + T cells (Tregs), and reduced stimulation of off-target effector T and NK cells. A library of rhIL-2 molecules was constructed with single site-specific, biorthogonal chemistry-compatible non-canonical amino acids installed near the interface where IL-2 engages its cognate receptor βγ (IL-2Rβγ) signaling complex. Biorthogonal site-specific pegylation and functional screening identified variants that retained engagement of the IL-2Rα chain with attenuated potency at the IL-2Rβγ complex.ResultsPhenotypic screening in mouse identifies SAR444336 (SAR’336; formerly known as THOR-809), rhIL-2 pegylated at H16, as a potential development candidate that specifically expands peripheral CD4+ Tregs with upregulation of markers that correlate with their suppressive function including FoxP3, ICOS and Helios, yet minimally expands CD8 + T or NK cells. In non-human primate, administration of SAR’336 also induces dose-dependent expansion of Tregs and upregulated suppressive markers without significant expansion of CD8 + T or NK cells. SAR’336 administration reduces inflammation in a delayed-type hypersensitivity mouse model, potently suppressing CD4+ and CD8 + T cell proliferation.ConclusionSAR’336 is a specific Treg activator, supporting its further development for the treatment of AI diseases.

Comparative analysis of the effects of cyclophosphamide and dexamethasone on intestinal immunity and microbiota in delayed hypersensitivity mice.

The T cell-mediated delayed-type hypersensitivity (DTH) response is critical for elucidating cellular immune mechanisms, especially the role of memory T cells upon antigen re-exposure. This study aimed to investigate the specific effects of the immunosuppressive drugs Cyclophosphamide (CY) and Dexamethasone (DEX) on intestinal immunity and microbiota in a DTH mouse model, contributing to a more nuanced understanding of their immunomodulatory mechanisms. Female BALB/c mice were sensitized to 2,4-dinitrofluorobenzene (DNFB) and randomly allocated into control, CY, and DEX groups. The impact of CY and DEX on immune function was assessed through measurement of thymus and spleen indices, lymphocyte proliferation in mesenteric lymph nodes (MLNs) using MTT assay, and flow cytometric analysis of T cell subsets and TCR expression. Intestinal secretory IgA (sIgA) was quantified by ELISA, and gut microbiota diversity was evaluated using 16S rRNA gene sequencing. CY and DEX significantly reduced the immune function in DNFB-induced sensitized mice, as indicated by decreased thymus and spleen indices, MLN enlargement, intestinal sIgA content, and ear swelling degree. Flow cytometry revealed that CY increased the proportion of total CD3+ T cells but reduced CD3+CD69+ activated T cells and CD3+TCRγ/δ+ T cells, while DEX increased CD3+CD4+ helper T cells. Both drugs induced distinct changes in gut microbiota diversity and structure, with CY enhancing α diversity and DEX reducing it. The study demonstrates that CY and DEX have distinct regulatory effects on the immune organ index, distribution of T cell subsets, and diversity and structure of gut microbiota on DTH-induced immune responses mice, suggesting their differential influence on intestinal mucosal immunity. These findings have implications for the development of targeted immunotherapies and understanding the interplay between immunosuppressive drugs and gut microbiota.

Sex differences in zymosan-induced behavioral visceral hypersensitivity and colorectal afferent sensitization.

Sex differences in visceral nociception have been reported in clinical and preclinical studies, but the potential differences in sensory neural encoding of the colorectum between males and females are not well understood. In this study, we systematically assessed sex differences in colorectal neural encoding by conducting high-throughput optical recordings in intact dorsal root ganglia (DRGs) from control and visceral hypersensitive mice. We found an apparent sex difference in zymosan-induced behavioral visceral hypersensitivity: enhanced visceromotor responses to colorectal distension were observed only in male mice, not in female mice. In addition, a higher number of mechanosensitive colorectal afferents were identified per mouse in the zymosan-treated male group than in the saline-treated male group, whereas the mechanosensitive afferents identified per mouse were comparable between the zymosan- and saline-treated female groups. The increased number of identified afferents in zymosan-treated male mice was predominantly from thoracolumbar (TL) innervation, which agrees with the significant increase in the TL afferent proportion in the zymosan group as compared with the control group in male mice. In contrast, female mice showed no difference in the proportion of colorectal neurons between saline- and zymosan-treated groups. Our results revealed a significant sex difference in colorectal afferent innervation and sensitization in the context of behavioral visceral hypersensitivity, which could drive differential clinical symptoms in male and female patients.NEW & NOTEWORTHY We used high-throughput GCaMP6f recordings to study 2,275 mechanosensitive colorectal afferents in mice. Our results revealed significant sex differences in the zymosan-induced behavioral visceral hypersensitivity, which were present in male but not female mice. Male mice also showed sensitization of colorectal afferents in the thoracolumbar pathway, whereas female mice did not. These findings highlight sex differences in sensory neural anatomy and function of the colorectum, with implications for sex-specific therapies for treating visceral pain.

Bivalent structure of a TNFR2-selective and agonistic TNF-α mutein Fc-fusion protein enhances the expansion activity of regulatory T cells

Recently, TNF receptor type 2 (TNFR2) signaling was found to be involved in the proliferation and activation of regulatory T cells (Tregs), a subpopulation of lymphocytes that suppress immune responses. Tregs mediate peripheral immune tolerance, and the disruption of their functions causes autoimmune diseases or allergy. Therefore, cell expanders or regulators of Tregs that control immunosuppressive activity can be used to treat these diseases. We focused on TNFR2, which is preferentially expressed on Tregs, and created tumor necrosis factor-α (TNF-α) muteins that selectively activate TNFR2 signaling in mice and humans, termed R2agoTNF and R2-7, respectively. In this study, we attempted to optimize the structure of muteins to enhance their TNFR2 agonistic activity and stability in vivo by IgG-Fc fusion following single-chain homo-trimerization. The fusion protein, scR2agoTNF-Fc, enhanced the expansion of CD4+CD25+ Tregs and CD4+Foxp3+ Tregs and contributed to their immunosuppressive activity ex vivo and in vivo in mice. The prophylactic administration of scR2agoTNF-Fc suppressed inflammation in contact hypersensitivity and arthritis mouse models. Furthermore, scR2-7-Fc preferentially expanded Tregs in human peripheral blood mononuclear cells via TNFR2. These TNFR2 agonist-Fc fusion proteins, which have bivalent structures, are novel Treg expanders.

CTLA-4 suppresses hapten-induced contact hypersensitivity in atopic dermatitis model mice

Atopic dermatitis (AD) patients with skin barrier dysfunction are considered to be at a higher risk of allergic contact dermatitis (ACD), although previous studies showed that attenuated ACD responses to strong sensitizers in AD patients compared to healthy controls. However, the mechanisms of ACD response attenuation in AD patients are unclear. Therefore, using the contact hypersensitivity (CHS) mouse model, this study explored the differences in CHS responses to hapten sensitization between NC/Nga mice with or without AD induction (i.e., non-AD and AD mice, respectively). In this study, ear swelling and hapten-specific T cell proliferation were significantly lower in AD than in non-AD mice. Moreover, we examined the T cells expressing cytotoxic T lymphocyte antigen-4 (CTLA-4), which is known to suppress T cell activation, and found a higher frequency of CTLA-4+ regulatory T cells in draining lymph node cells in AD than in non-AD mice. Furthermore, the blockade of CTLA-4 using a monoclonal antibody eliminated the difference in ear swelling between non-AD and AD mice. These findings suggested that CTLA-4+T cells may contribute to suppressing the CHS responses in AD mice.

The effects of cannabinoid 1 receptor blockade on oxazolone-induced contact hypersensitivity mouse model

Abstract Contact hypersensitivity (CHS) is an experimental model of human allergic contact dermatitis, one of the representative skin diseases, where NLRP3 inflammasome plays a crucial role. Although the regulation of NLRP3 inflammasome activity by cannabinoid 1 receptor (CB1R) has been shown in metabolic diseases such as obesity and type 2 diabetes, the role of CB1R in CHS is unclear. Here, we investigate the putative role of CB1R in CHS by using a selective CB1R antagonist rimonabant. Pharmacological blockade of CB1R by rimonabant ameliorated oxazolone-induced CHS by suppressing hyperproliferation of keratinocytes and expression of pro-inflammatory cytokines in a TLR2/4-dependent manner. The inhibitory effect of rimonabant in oxazolone-induced CHS was not observed in NLRP3-deficient mice. These results suggest that CB1R could control TLR2/4 signaling pathway via NLRP3 inflammasome in CHS. Supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education (NRF-2022R1I1A1A01071661)

146 miR-31 and IL-34 expression are regulated in response to DNFB in the contact hypersensitivity mouse model

146 miR-31 and IL-34 expression are regulated in response to DNFB in the contact hypersensitivity mouse model

Dihydroartemisinin-ursodeoxycholic acid conjugate is a potential treatment agent for inflammatory bowel disease.

A novel artemisinin derivative, dihydroartemisinin-ursodeoxycholic acid conjugate (4), was found to exhibit strong immunosuppressive activity. Various methods were used to evaluate the immunosuppressive activity and mechanism of action of the compound to explore its potential applications. T cell proliferation, mixed lymphocyte reaction (MLR), and Th1/Th17 differentiation assays were used to evaluate the immunosuppressive activity of the compound. Differentially expressed genes from RNA sequencing were analysed with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, while enriched signalling pathways were further validated by western blotting (WB). In vivo efficacy was validated with delayed-type hypersensitivity (DTH) mouse models and dextran sodium sulphate (DSS)-induced inflammatory bowel disease (IBD) mouse model. Compound 4 inhibited concanavalin A -induced mouse splenic T cell proliferation (IC50=15nM) and anti-CD3/CD28-induced human primary T cell proliferation (IC50=30nM) while also reducing the secretion of hIFN-γ. Compound 4 exhibited similar inhibitory activity in MLR assay. Compound 4 dose-dependently inhibited human Th1/Th17 differentiation. The KEGG pathway enrichment analysis indicated that the genes related to T cell activation signalling pathways PI3K-AKT, MAPK, and NF-κB were significantly enriched. WB confirmed that compound 4 inhibited the AKT/MAPK and NF-κB signalling pathways. Compound 4 dose-dependently inhibited ear and foot pad swelling in DTH mouse models. In the DSS-induced IBD mouse model, compound 4 significantly decreased the disease activity index and colon density, and inhibited splenomegaly of the mice. The in vitro and in vivo results indicated that compound 4 has the potential to be developed into an anti-IBD drug.

Melittin-Carrying Nanoparticle Suppress T Cell-Driven Immunity in a Murine Allergic Dermatitis Model.

Allergic contact dermatitis (ACD) and atopic dermatitis (AD) are the most common human skin disorders. Although corticosteroids have been widely used to treat ACD and AD, the side effects of corticosteroids encourage researchers to explore new immunoregulatory treatments. Here, an immunomodulatory approach based on lipid nanoparticles carrying α-helical configurational melittin (α-melittin-NP) is developed to overcome T cell-mediated inflammatory reactions in an oxazolone (OXA)-induced contact hypersensitivity mouse model and OXA-induced AD-like mouse model. Intradermal injection of low-dose α-melittin-NPs prevents the skin damage caused by melittin administration alone and efficiently targeted lymph nodes. Importantly, melittin and α-melittin-NPs restrain RelB activity in dendritic cells (DCs) and further suppresses dendritic cell activation and maturation in lymph nodes. Furthermore, low-dose α-melittin-NPs leads to relief of antigen recognition-induced effector T cell arrest in the dermis and inhibited allergen-specific T cell proliferation and activation. Significantly, this approach successfully controls Th1-type cytokine release in the ACD model and restricts Th2-type cytokine and IgE release in the AD-like model. Overall, intradermal delivery of low-dose α-melittin-NPs efficiently elicits immunosuppression against T cell-mediated immune reactions, providing a promising therapeutic strategy for treating skin disorders not restricted to the lesion region.

Nanoparticle dose and antigen loading attenuate antigen-specific T-cell responses.

Immune-mediated hypersensitivities such as autoimmunity, allergy, and allogeneic graft rejection are treated with therapeutics that suppress the immune system, and the lack of specificity is associated with significant side effects. The delivery of disease-relevant antigens (Ags) by carrier systems such as poly(lactide-co-glycolide) nanoparticles (PLG-Ag) and carbodiimide (ECDI)-fixed splenocytes (SP-Ag) has demonstrated Ag-specific tolerance induction in model systems of these diseases. Despite therapeutic outcomes by both platforms, tolerance is conferred with different efficacy. This investigation evaluated Ag loading and total particle dose of PLG-Ag on Ag presentation in a coculture system of dendritic cells (DCs) and Ag-restricted T cells, with SP-Ag employed as a control. CD25 expression was observed in nearly all T cells even at low concentrations of PLG-Ag, indicating efficient presentation of Ag by dendritic cells. However, the secretion of IL-2, Th1, and Th2 cytokines (IFNγ and IL-4, respectively) varied depending on PLG-Ag concentration and Ag loading. Concentration escalation of soluble Ag resulted in an increase in IL-2 and IFNγ and a decrease in IL-4. Treatment with PLG-Ag followed a similar trend but with lower levels of IL-2 and IFNγ secreted. Transcriptional Activity CEll ARrays (TRACER) were employed to measure the real-time transcription factor (TF) activity in Ag-presenting DCs. The kinetics and magnitude of TF activity was dependent on the Ag delivery method, concentration, and Ag loading. Ag positively regulated IRF1 activity and, as carriers, NPs and ECDI-treated SP negatively regulated this signaling. The effect of Ag loading and dose on tolerance induction were corroborated in vivo using the delayed-type hypersensitivity (DTH) and experimental autoimmune encephalomyelitis (EAE) mouse models where a threshold of 8 μg/mg Ag loading and 0.5 mg PLG-Ag dose were required for tolerance. Together, the effect of Ag loading and dosing on in vitro and in vivo immune regulation provide useful insights for translating Ag-carrier systems for the clinical treatment of immune disorders.

Skin microbiota of oxazolone-induced contact hypersensitivity mouse model.

Contact allergy is a common skin allergy, which can be studied utilising contact hypersensitivity (CHS) animal model. However, it is not clear, whether CHS is a suitable model to investigate skin microbiota interactions. We characterised the effect of contact dermatitis on the skin microbiota and studied the biological effects of oxazolone (OXA) -induced inflammation on skin thickness, immune cell numbers and changes of the microbiota in CHS mouse model (n = 72) for 28 days. Through 16S rRNA gene sequencing we defined the composition of bacterial communities and associations of bacteria with inflammation. We observed that the vehicle solution of acetone and olive oil induced bacterial community changes on day 1, and OXA-induced changes were observed mainly on day 7. Many of the notably enriched bacteria present in the OXA-challenged positive group represented the genus Faecalibaculum which were most likely derived from the cage environment. Additionally, skin inflammation correlated negatively with Streptococcus, which is considered a native skin bacterium, and positively with Muribacter muris, which is typical in oral environment. Skin inflammation favoured colonisation of cage-derived faecal bacteria, and additionally mouse grooming transferred oral bacteria on the skin. Due to the observed changes, we conclude that CHS model could be used for certain skin microbiome-related research set-ups. However, since vehicle exposure can alter the skin microbiome as such, future studies should include considerations such as careful control sampling and statistical tests to account for potential confounding factors.

Cannabigerol (CBG) attenuates mechanical hypersensitivity elicited by chemotherapy-induced peripheral neuropathy.

Cannabigerol (CBG) is a non-psychoactive phytocannabinoid produced by the plant Cannabis sativa with affinity to various receptors involved in nociception. As a result, CBG is marketed as an over-the-counter treatment for many forms of pain. However, there is very little research-based evidence for the efficacy of CBG as an anti-nociceptive agent. To begin to fill this knowledge gap, we assessed the anti-nociceptive effects of CBG in C57BL/6 mice using three different models of pain; cisplatin-induced peripheral neuropathy, the formalin test, and the tail-flick assay. Using the von Frey test, we found that CBG-attenuated mechanical hypersensitivity evoked by cisplatin-induced peripheral neuropathy in both male and female mice. Additionally, we observed that this CBG-induced reduction in mechanical hypersensitivity was attenuated by the α2 -adrenergic receptor antagonist atipamezole (3mg/kg, i.p.) and the CB1 R antagonist, AM4113 (3mg/kg, i.p.), and blocked by the CB2 R antagonist/inverse agonist, SR144528 (10mg/kg, i.p.). We found that the TRPV1 antagonist, SB705498 (20 mg/kg, i.p.) was unable to prevent CBG actions. Furthermore, we show that CBG:CBD oil (10mg/kg, i.p.) was more effective than pure CBG (10mg/kg) at reducing mechanical hypersensitivity in neuropathic mice. Lastly, we show that pure CBG and CBG:CBD oil were ineffective at reducing nociception in other models of pain, including the formalin and tail flick assays. Our findings support the role of CBG in alleviating mechanical hypersensitivity evoked by cisplatin-induced peripheral neuropathy, but highlight that these effects may be limited to specific types of pain. There are few effective treatments for neuropathic pain and neuropathic pain is projected to increase with the aging population. We demonstrate that CBG (cannabigerol) and CBG:CBD oil attenuate neuropathy-induced mechanical hypersensitivity mice. Second, we identify receptor targets that mediate CBG-induced reduction in mechanical hypersensitivity in neuropathic mice. Third, we demonstrate that an acute injection of CBG is anti-nociceptive specifically for neuropathic pain rather than other forms of pain, including persistent pain and thermal pain.

Unveiling the improved targeting migration of mesenchymal stem cells with CXC chemokine receptor 3-modification using intravital NIR-II photoacoustic imaging

BackgroundTherapy with genetically modified mesenchymal stem cells (MSCs) has clinical translation promise. Optimizing the targeting migratory ability of MSCs relies on accurate imaging of the distribution and extravasation kinetics of MSCs, and the corresponding imaging results could be used to predict therapeutic outcomes and guide the optimization of the treatment program. Among the different imaging modalities, second near-infrared (NIR-II) optical-resolution photoacoustic microscopy (OR-PAM) has merits, including a fine resolution, a deep penetration, a high sensitivity, and a large signal-to-background ratio. It would be an ideal candidate for precise monitoring of MSCs, although it has not been tested for this purpose so far.ResultsPenetrating peptide-decorated conjugated polymer nanoparticles (TAT-CPNPs) with strong NIR-II absorbance were used to label chemokine-receptor genetically modified MSCs, which were subsequently evaluated under intravital NIR-II OR-PAM regarding their targeting migratory ability. Based on the upregulation of chemokine (C-X-C motif) ligand 10 in the inflamed ears of contact hypersensitivity mice, MSCs with overexpression of corresponding receptor, chemokine (C-X-C motif) receptor 3 (Cxcr3) were successfully generated (MSCCxcr3). TAT-CPNPs labeling enabled NIR-II photoacoustic imaging to discern MSCCxcr3 covered by 1.2 cm of chicken breast tissue. Longitudinal OR-PAM imaging revealed enhanced inflammation-targeting migration of MSCCxcr3 over time attributed to Cxcr3 gene modification, which was further validated by histological analysis.ConclusionsTAT-CPNPs-assisted NIR-II PA imaging is promising for monitoring distribution and extravasation kinetics of MSCs, which would greatly facilitate optimizing MSC-based therapy.Graphical

Identification and quantification of nociceptive Schwann cells in mice with and without Streptozotocin-induced diabetes

Specialized cutaneous Schwann cells (SCs), termed nociceptive SCs, were recently discovered. Their function is not fully understood, but they are believed not only to support peripheral axons in mouse skin by forming a mesh-like neural-glio networking structure in subepidermal area, but also contributing to transduction of mechanical sensation and neuropathic pain. Diabetic neuropathy (DPN) is one of the most common complication of diabetes, however, the mechanisms behind painful and painless DPN remain unclear. Using a mouse model of DPN, we want to investigate if there are quantitative differences in nociceptive SC density between the condition of hyperglycemia-induced sensory abnormalities and control condition and at which stage in the disease the damage occurs. Here, we developed a set of counting rules for nociceptive SCs based on immunofluorescent staining, and applied the method to quantify the density of nociceptive SCs in control mice (n = 10), mice with nociceptive hypersensitivity at early diabetic stage (n = 5), and mice with sensory hyposensitivity at late diabetic stage (n = 5) in the Streptozotocin (STZ) model of type 1 diabetes. Nociceptive SCs were identified as S100+/Sox10+/DAPI+ cells abutting to peripheral nerves, with the somas located within 25 µm depth in the subepidermal area and outside glands and large fiber bundles. Hypersensitive diabetic mice had decreased nociceptive SC density, despite having normal epidermal nerve fiber density, compared with age-matched control mice (P = 0.023). In contrast, there was a reduction in intraepidermal nerve fiber density but no difference in nociceptive SC density between hyposensitive diabetic mice and the age-matched control mice. This study provides a detailed description of how to identify and quantify nociceptive SC and demonstrates that nociceptive SC density declines before nerve fiber deterioration, which supports previous observations that nociceptive SCs are critical for maintenance of cutaneous sensory nerves.

SPARC: Sex difference in colorectal afferent sensitization in zymosan‐induced visceral hypersensitivity as revealed by high‐throughput GCaMP recordings in thoracolumbar and lumbosacral DRG

Female patients with irritable bowel syndrome (IBS) report more intense visceral pain sensation than male patients. Female rats show more sensitive and cycle‐dependent visceromotor responses (VMR) to graded colorectal distension (CRD). Both clinical psychophysical evidence and preclinical behavioral data support an apparent sex difference in visceral nociception. However, very little is known regarding the potential differences in sensory neural encoding of distal colon and rectum (colorectum) between males and females. In this study, we systematically assessed the sex difference in colorectal neural encoding by conducting high‐throughput optical recordings in intact dorsal root ganglia (DRG) from control and visceral hypersensitive mice. We crossbred GCaMP6f mice with VGLUT2‐Cre mice to express GCamP6f gene in most colorectal sensory neurons. Mice receiving intracolonic treatment of zymosan (30mg/mL, 0.2mL) for three consecutive days developed behavioral visceral hypersensitivity as validated by enhanced VMR to CRD. We then harvested the colorectum with pelvic nerve, lumbar splanchnic nerve and T12 to S1 DRG in continuity in an ex vivo preparation for GCaMP6f recordings. A total of 2275 colorectal afferents were characterized from both sexes in control and zymosan‐treated groups. DRG neurons responding to either colorectal distension (15, 30, 45 and 60 mmHg) and/or mucosal shearing (20‐30 mL/min) were functionally categorized into four classes: mucosal, muscular‐mucosal and high‐ and low‐threshold muscular afferents. In control group, the number of colorectal afferents recorded per mouse were slightly lower in male mice than in female ones (38.7 vs. 45.2) with no significant difference in their distributions across thoracolumbar (TL) and lumbosacral (LS) DRG. In zymosan group, more afferents were recorded from each male mice than female ones (53.8 vs. 44.1) showing significantly higher TL innervation (T12, L1, and L2 DRG) in male group. Within the male groups, zymosan treatment resulted in a significant increase in the proportion of TL innervation as compared with saline treatment. In contrast, female mice showed no difference in the proportion of colorectal neurons between saline‐ and zymosan‐treated groups. Our results have revealed a significant sex difference in colorectal afferent innervation and sensitization in the context of behavioral visceral hypersensitivity. The current outcomes draw further focused research on the neurophysiological differences between sexes that could drive the differential gastrointestinal symptoms in male and female IBS patients.

Alternative Splicing of Neuropeptide Prohormone and Receptor Genes Associated with Pain Sensitivity Was Detected with Zero-Inflated Models.

Migraine is often accompanied by exacerbated sensitivity to stimuli and pain associated with alternative splicing of genes in signaling pathways. Complementary analyses of alternative splicing of neuropeptide prohormone and receptor genes involved in cell–cell communication in the trigeminal ganglia and nucleus accumbens regions of mice presenting nitroglycerin-elicited hypersensitivity and control mice were conducted. De novo sequence assembly detected 540 isoforms from 168 neuropeptide prohormone and receptor genes. A zero-inflated negative binomial model that accommodates for potential excess of zero isoform counts enabled the detection of 27, 202, and 12 differentially expressed isoforms associated with hypersensitivity, regions, and the interaction between hypersensitivity and regions, respectively. Skipped exons and alternative 3′ splice sites were the most frequent splicing events detected in the genes studied. Significant differential splicing associated with hypersensitivity was identified in CALCA and VGF neuropeptide prohormone genes and ADCYAP1R1, CRHR2, and IGF1R neuropeptide receptor genes. The prevalent region effect on differential isoform levels (202 isoforms) and alternative splicing (82 events) were consistent with the distinct splicing known to differentiate central nervous structures. Our findings highlight the changes in alternative splicing in neuropeptide prohormone and receptor genes associated with hypersensitivity to pain and the necessity to target isoform profiles for enhanced understanding and treatment of associated disorders such as migraine.

Olorinab (APD371), a peripherally acting, highly selective, full agonist of the cannabinoid receptor 2, reduces colitis-induced acute and chronic visceral hypersensitivity in rodents.

Abdominal pain is a key symptom of inflammatory bowel disease and irritable bowel syndrome, for which there are inadequate therapeutic options. We tested whether olorinab-a highly selective, full agonist of the cannabinoid receptor 2 (CB2)-reduced visceral hypersensitivity in models of colitis and chronic visceral hypersensitivity (CVH). In rodents, colitis was induced by intrarectal administration of nitrobenzene sulfonic acid derivatives. Control or colitis animals were administered vehicle or olorinab (3 or 30 mg/kg) twice daily by oral gavage for 5 days, starting 1 day before colitis induction. Chronic visceral hypersensitivity mice were administered olorinab (1, 3, 10, or 30 mg/kg) twice daily by oral gavage for 5 days, starting 24 days after colitis induction. Visceral mechanosensitivity was assessed in vivo by quantifying visceromotor responses (VMRs) to colorectal distension. Ex vivo afferent recordings determined colonic nociceptor firing evoked by mechanical stimuli. Colitis and CVH animals displayed significantly elevated VMRs to colorectal distension and colonic nociceptor hypersensitivity. Olorinab treatment significantly reduced VMRs to control levels in colitis and CVH animals. In addition, olorinab reduced nociceptor hypersensitivity in colitis and CVH states in a concentration- and CB2-dependent manner. By contrast, olorinab did not alter VMRs nor nociceptor responsiveness in control animals. Cannabinoid receptor 2 mRNA was detected in colonic tissue, particularly within epithelial cells, and dorsal root ganglia, with no significant differences between healthy, colitis, and CVH states. These results demonstrate that olorinab reduces visceral hypersensitivity through CB2 agonism in animal models, suggesting that olorinab may provide a novel therapy for inflammatory bowel disease- and irritable bowel syndrome-associated abdominal pain.