Evaluation of morphine analgesia and motor coordination in mice following cortex-specific knockout of the N-methyl-D-aspartate NR1-subunit

Studies have shown that N-methyl-d-aspartate (NMDA) receptors play a critical role in morphine analgesia and motoric processes at different levels of the central nervous system. In this study, we used cortex-specific NR1 knockout (KO) mice (C57BL/6 strain) to elucidate the role of cortical NMDA receptors in morphine analgesia and motor coordination. On post-natal day 20, mice (CTL and KO) received vehicle (saline) or morphine (10mg/kg) and paw withdrawal latency (PWL) to a noxious thermal stimulus was measured. On post-natal day 21, motor coordination was measured using the rotating pole test. No differences in KO mice were found with respect to PWL following administration of saline or morphine (p>0.05). However, sex-dependent differences were found in motor coordination, with male KO mice showing a greater motor impairment in the rotating pole test than female KO mice (p<0.05). The present results demonstrate that NMDA receptors are involved in both the analgesic effects of morphine and motor coordination, with the existence of sex-related differences in motor coordination.     

Deficiency of Vlgr1 resulted in deafness and susceptibility to audiogenic seizures while the degree of hearing impairment was not correlated with seizure severity in C57BL/6- and 129-backcrossed lines of Vlgr1 knockout mice

Vlgr1 (very large G-protein coupled receptor 1) knockout mice against hybrid backgrounds of the 129/Ola and C57BL/6 mouse strains show hearing deficit and high susceptibility to audiogenic seizures. The present study examined how hearing impairment and susceptibility to audiogenic seizures in Vlgr1-deficient mice change according to the genetic background of 129 and C57BL/6 mouse strains, which are popular strains for genetic studies. C57BL/6 mice have normal hearing ability during adolescence and are resistant to audiogenic seizures, and the 129S1/SvImJ substrain does not have a severe hearing deficit or convulsions as a result of audiogenic seizures; therefore, these strains were chosen for the present backcross study. C57BL/6-backcrossed Vlgr1 knockout mice and 129 (129S1/SvImJ)-backcrossed Vlgr1 knockout mice were established and their phenotypes investigated. Vlgr1 knockout mice showed hearing loss and high susceptibility to audiogenic seizures regardless of their genetic backgrounds. 129-backcrossed Vlgr1 knockout mice exhibited 10–20 dB more severe hearing loss than C57BL/6-backcrossed Vlgr1 knockout mice. In general, 129-backcrossed Vlgr1 knockout mice showed a higher incidence of wild running than C57BL/6-backcrossed Vlgr1 knockout mice, and this incidence became smaller as they matured. However, C57BL/6-backcrossed Vlgr1 knockout mice showed a significantly higher mortality rate as a result of auditory stimulation 3 weeks postnatally than 129-backcrossed mice.     

Genetic deletion of the adenosine A2A receptor in mice reduces the changes in spinal cord NMDA receptor binding and glucose uptake caused by a nociceptive stimulus

Mice lacking the adenosine A_2A receptor are less sensitive to nociceptive stimuli, and A_2A receptor antagonists have antinociceptive effects. We have previously shown a marked reduction in the behavioural responses to formalin injection in A_2A receptor knockout mice. This may be due to the presence of pronociceptive A_2A receptors on sensory nerves, and if so spinal cords from A_2A receptor knockout mice may have altered neurochemical responses to a nociceptive stimulus. We tested this hypothesis by studying two parameters known to change with spinal cord activity, NMDA glutamate receptor binding and [¹⁴C]-2-deoxyglucose uptake, following intraplantar formalin injection in wild-type and A_2A receptor knockout mice. In naïve untreated A_2A knockout mice [¹⁴C]-2-deoxyglucose uptake in all regions of the spinal cord was significantly lower compared to the wild-type, similar to the reduced NMDA receptor binding that we have previously observed. Following formalin treatment, there was an decrease in [³H]-MK801 binding to NMDA receptors and an increase in [¹⁴C]-2-deoxyglucose uptake in the spinal cords of wild-type mice, and these changes were significantly reduced in the A_2A knockout mice. In addition to altered behavioural responses, there are therefore corresponding reductions in spinal cord neurochemical changes induced by formalin in mice lacking adenosine A_2A receptors. These observations support the hypothesis that activation of A_2A receptors enhances nociceptive input into the spinal cord and suggests a possible role for A_2A antagonists as analgesics.     

Strain-dependent requirement for IFN-γ for respiratory control and immunotherapy in murine gammaherpesvirus infection

Interferon-γ (IFN-γ) and perforin (pfp) are important effector mechanisms used by CD8 T cells to clear virus-infected cells. In this study, we used IFN-γ/pfp double knockout mice to address if these two effector molecules play redundant roles in the control of acute infection with murine gammaherpesvirus-68 (MHV-68) in BALB/C mice. Perforin knockout (KO) mice and wild-type mice cleared infectious virus from the lungs, even following high-dose infection. However, the IFN-γ KO and IFN-γ/pfp double knockout (DKO) groups had higher virus titers in the lungs at day 10 post-infection, and both groups had higher mortality rates. In IFN-γ/pfp DKO mice, the virus titer and mortality rate were significant higher than in IFN-γ KO mice, indicating a role for perforin in protection from disease. WT mice given IFN-γ blocking antibody also showed significantly higher viral titers. In contrast, IFN-γ KO mice on a C57BL/6 background controlled respiratory infection comparably to wild-type mice. These data show that perforin plays a redundant role in the control of virus replication, but IFN-γ plays an essential role in BALB/C mice infected with MHV-68. We conclude that there is a marked strain-dependent difference in the effector mechanisms needed to control acute MHV-68 infection between C57BL/6 and BALB/C mice. In addition we show that immune therapy that re-establishes viral control after spontaneous reactivation in CD4-deficient mice depends upon perforin in C57BL/6 mice but IFN-γ in BALB/C mice.     

C3 is central to the interstitial component of experimental immune complex glomerulonephritis

We have suggested that renal tubular synthesis of C3 and its activation in the cortical interstitium is a mechanism for the progression of glomerulonephritis to interstitial injury. To test this hypothesis, immune complex glomerulonephritis was induced in C57BL/6 mice by intraperitoneal injections of horse spleen apoferritin and lipopolysaccharide (HSA/LPS). When compared to wild-type (WT) animals, C3 knockout (C3KO) mice had glomerular changes that were identical. Morphometric analysis of the cortical interstitium, however, showed marked differences. WT mice had more interstitial inflammation, edema, and tubular atrophy, when compared to C3KO mice. At the end of the experiment, WT animals also had significantly more proteinuria than did C3KOs. These experiments provide further evidence of a role of locally synthesized complement in the progression of glomerular disease.     

Temperature-dependent N-methyl-d-aspartate receptor-mediated cytotoxicity in cultured rat cortical neurons

Hypothermia protects against hypoxic or ischemic damage. However, the mechanisms by which brain cooling prevents hypoxic or ischemic damage are not clear. We examined whether hypothermia protects against excitotoxicity in cultured cortical cells. Exposure of cortical cell culture to 500 μM N-methyl-d-aspartate (NMDA) for 15 min at 32 °C or 37 °C did not induce neurotoxicity. On the other hand, reduction of temperature to 20 °C resulted in widespread neuronal disintegration by the following day. Moreover, intracellular calcium concentration increased markedly by adding NMDA to cells at 20 °C. These results suggest that profound hypothermia does not protect neurons from excitotoxicity by inhibiting NMDA receptor activity.     

Characteristic pattern of skeletal muscle remodelling in different mouse strains

Muscular injury associated with local inflammatory reaction frequently occurs in sports medicine, but the individual response and capacity of regeneration vary among subjects. Inflammatory cytokines are probably implicated in activation of repair mechanisms by specifically influencing tissue microenvironment. This work aimed to compare muscle tissue repair in different mouse lineages. We used C57BL/6 and BALB/c mice genetically predisposed to either Type1 or Type2 cytokine production. The role of Type1 cytokines was also investigated in C57IFN‐γ (IFNγ‐KO) and C57IL‐12 (IL12‐KO) knockout mice. Participation of T lymphocytes was assessed in athymic BALB/c nude (nu/nu) mice. Muscular lesion was induced with bupivacaine injection in the Triceps brachii muscle. BALB/c mice showed marked collagen deposition and increased TGF‐β mRNA content, contrasting with mild fibrosis observed in C57BL/6 mice. C57‐IFNγ‐KO mice, exhibited pronounced fibrosis, but IL12‐KO collagen deposition was similar to that of C57. Twenty‐four hours after lesion, C57BL/6 and BALB/c^nu/nu presented numerous regenerating myofibres and marked increase of metalloprotease‐9 activity compared with BALB/c. These data support that skeletal muscle remodelling is greatly influenced by the genetic backgrounds, shedding light on the molecular mechanisms influencing differential muscular remodelling and tissue regeneration among individuals.     

Lactobacillus rhamnosus Ingestion Promotes Innate Host Defense in an Enteric Parasitic Infection

Enteric parasite infections around the world are a huge economic burden and decrease the quality of life for many people. The use of beneficial bacteria has attracted attention for their potential therapeutic applications in various diseases. However, the effects of beneficial bacteria in enteric parasitic infections remain largely unexplored. We investigated the effects of ingestion of Lactobacillus rhamnosus (JB-1) in a model of enteric nematode (Trichuris muris) infection. C57BL/6 (resistant to infection), AKR (susceptible to infection), interleukin 10 (IL-10) knockout (KO), and mucin Muc2 KO mice were infected with T. muris and treated orally with probiotic JB-1 or medium. The mice were sacrificed on various days postinfection to examine goblet cells, epithelial cell proliferation, cytokines, and worm burdens. Treatment with JB-1 significantly enhanced worm expulsion in resistant C57BL/6 mice, and this was associated with increases in IL-10 levels, goblet cell numbers, and epithelial cell proliferation. Beneficial effects of JB-1 were absent in IL-10 KO and resistant mice treated with γ-irradiated bacteria. Live JB-1 treatment also expedited worm expulsion in Muc2 KO mice and, more importantly, in AKR mice (susceptible to infection). Injection of IL-10 directly into the colonic tissue of uninfected mice induced goblet cell hyperplasia. These findings demonstrate that JB-1 modulates goblet cell biology and promotes parasite expulsion via an IL-10-mediated pathway and provide novel insights into probiotic effects on innate defense in nematode infection.     

Deletion of the GluR5 subunit of kainate receptors affects cocaine sensitivity and preference

We have demonstrated previously that mice expressing a constitutive deletion of the kainate receptor subunit GluR5 (GluR5 KO) do not differ from wildtype (WT) littermates of a congenic C57BL/6 background with regard to both the development of morphine physical dependence as measured by naloxone-precipitated withdrawal signs and to morphine reward as revealed by the expression of conditioned place preference (CPP). However, unlike WT, GluR5 KO mice fail to develop antinociceptive tolerance following repeated systemic morphine administration. In this report, we examined the impact of GluR5 deletion on cocaine-mediated CPP and locomotor sensitization. Expression of CPP was evident in WT mice following repeated daily administration of 20 mg/kg (but not 10 mg/kg) i.p. cocaine. Interestingly, GluR5 KO mice exhibited enhanced cocaine preference as compared with WT mice at both 10 and 20 mg/kg doses. In addition, while GluR5 KO mice did not differ from WT with respect to baseline locomotor activity, mutant mice demonstrated increased locomotor hyperactivity versus WT mice after repeated injection of 15 mg/kg i.p. cocaine. Collectively, these data indicate that GluR5 appears to negatively modulate some psychostimulant and rewarding properties of cocaine, as demonstrated by heightened sensitization and salience in mutant mice.     

Spinal N-methyl-D-aspartate receptors and nociception-evoked release of primary afferent substance P

Dorsal horn N-methyl-D-aspartate (NMDA) receptors contribute significantly to spinal nociceptive processing through an effect postsynaptic to non-primary glutamatergic axons, and perhaps presynaptic to the primary afferent terminals. The present study sought to examine the regulatory effects of NMDA receptors on primary afferent release of substance P (SP), as measured by neurokinin 1 receptor (NK1r) internalization in the spinal dorsal horn of rats. The effects of intrathecal NMDA alone or in combination with D-serine (a glycine site agonist) were initially examined on basal levels of NK1r internalization. NMDA alone or when co-administered with D-serine failed to induce NK1r internalization, whereas activation of spinal TRPV1 receptors by capsaicin resulted in a notable NK1r internalization. To determine whether NMDA receptor activation could potentiate NK1r internalization or pain behavior induced by a peripheral noxious stimulus, intrathecal NMDA was given prior to an intraplantar injection of formalin. NMDA did not alter the formalin-induced NK1r internalization nor did it enhance the formalin paw flinching behavior. To further characterize the effects of presynaptic NMDA receptors, the NMDA antagonists DL-2-amino-5-phosphonopentanoic acid (AP-5) and MK-801 were intrathecally administered to assess their regulatory effects on formalin-induced NK1r internalization and pain behavior. AP-5 had no effect on formalin-induced NK1r internalization, whereas MK-801 produced only a modest reduction. Both antagonists, however, reduced the formalin paw flinching behavior. In subsequent in vitro experiments, perfusion of NMDA in spinal cord slice preparations did not evoke basal release of SP or calcitonin gene-related peptide (CGRP). Likewise, perfusion of NMDA did not enhance capsaicin-evoked release of the two peptides. These results suggest that presynaptic NMDA receptors in the spinal cord play little if any role on the primary afferent release of SP.     

Endogenous pain modulation during the formalin test in estrogen receptor beta knockout mice

The involvement of estrogen in pain has been investigated in many ways. However the specific role played by estrogen receptors remains elusive. Estrogen receptors alpha and beta mediate different physiological functions. For example, estrogen receptor beta is more closely related to non-reproductive effects than the alpha subtype is. To verify the involvement of estrogen receptor beta on acute and persistent pain as well as on endogenous pain inhibitory mechanisms, hotplate and formalin tests were carried out in wild type (WT) and estrogen receptor beta knockout (ERbeta KO) mice of both sexes. Ovariectomies followed by estrogen and progesterone replacement were performed in female groups to insure comparable sex hormone levels. We found that nociceptive responses are lower in ERbeta KO female than in WT female mice during the interphase and early tonic phase II of the formalin test but not during acute and late tonic phases. Moreover, behavioral and spinal (c-Fos) differences were only observed in females. ERbeta KO females had lower c-Fos expression in laminae I-II and IV-V of the spinal cord than WT females. These results suggest that estrogen, through its actions on ERbeta, dampens the efficacy of endogenous pain modulation mechanisms during the interphase and/or inflammation process in the early phase II, triggering an increase in spinal nociceptive neuronal activity. This confirms our previous observations that estrogen specifically influences nociceptive responses during the interphase of the formalin test and demonstrates a role for ERbeta on endogenous pain modulation systems.     

Melanocortin-1 receptor gene variants affect pain and mu-opioid analgesia in mice and humans

Background: A recent genetic study in mice and humans revealed the modulatory effect of MC1R (melanocortin-1 receptor) gene variants on κ-opioid receptor mediated analgesia. It is unclear whether this gene affects basal pain sensitivity or the efficacy of analgesics acting at the more clinically relevant µ-opioid receptor.

Objective: To characterise sensitivity to pain and µ-opioid analgesia in mice and humans with non-functional melanocortin-1 receptors.

Methods: Comparisons of spontaneous mutant C57BL/6-Mc1r^e/e mice to C57BL/6 wildtype mice, followed by a gene dosage study of pain and morphine-6-glucuronide (M6G) analgesia in humans with MC1R variants.

Results: C57BL/6-Mc1r^e/e mutant mice and human redheads—both with non-functional MC1Rs—display reduced sensitivity to noxious stimuli and increased analgesic responsiveness to the µ-opioid selective morphine metabolite, M6G. In both species the differential analgesia is likely due to pharmacodynamic factors, as plasma levels of M6G are similar across genotype.

Conclusions: Genotype at MC1R similarly affects pain sensitivity and M6G analgesia in mice and humans. These findings confirm the utility of cross species translational strategies in pharmacogenetics.

     

利用CRISPR/Cas9技术制备成对框基因2敲除小鼠

目的 利用成簇规律间隔短回文重复序列（CRISPR）/重组CRISPR相关核酸酶9（Cas9）技术双切口法制备成对框基因2（Pax2）敲除小鼠,为探讨Pax2基因在多个系统发育的作用提供动物模型。方法 根据Pax2基因序列设计sgRNA,设计出的sgRNA和Cas9体外转录后显微注射到C57BL/6J 小鼠的受精卵中,F0代小鼠出生后取其基因DNA测序鉴定基因型。共获得8只F0 代小鼠,使敲除成功的F0代小鼠与野生C57BL/6J 小鼠交配,获得F1代小鼠,后均采用基因成功敲除的小鼠与C57BL/6J 小鼠进行交配,可获得稳定的Pax2基因敲除小鼠。结果 成功获得可稳定繁殖的Pax2杂合子基因敲除小鼠,其Pax2基因缺失1628 bp;组织HE染色显示,敲除小鼠的肾小球数量明显减少;Western blotting结果显示,敲除小鼠的肾皮质Pax2蛋白表达较野生型小鼠减少。结论 利用CRISPR/Cas9技术可成功构建Pax2杂合子基因敲除小鼠,为进一步研究Pax2基因的作用奠定基础。     

Differential effects of NMDA and AMPA/KA receptor antagonists on c-Fos or Zif/268 expression in the rat spinal dorsal horn induced by noxious thermal or mechanical stimulation,or formalin injection

The involvement of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate (KA) receptors in the induction of c-Fos and Zif/268 expression in spinal dorsal horn neurons following noxious thermal or mechanical stimulation, or formalin injection into the rat hind paw was examined by intrathecal administration of a competitive NMDA receptor antagonist, 2-amino-5-phosphonopentanoic acid (APV) or an AMPA/KA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), or both, 30 min prior to noxious stimulation. APV caused a significant reduction in the level of c-Fos expression in the superficial layer induced by each of these three noxious stimuli. The effects of APV on Zif/268 expression or of CNQX on c-Fos or Zif/268 expression in the superficial layer induced by these three noxious stimuli were dependent on the type of stimulus applied to the rat hind paw. The noxious thermal stimulus-evoked c-Fos expression level was reduced by APV and/or CNQX, while Zif/268 expression was hardly changed. Both c-Fos and Zif/268 expressions following formalin injection were reduced by APV alone and APV+CNQX, but not by CNQX alone. Zif/268 expression following noxious mechanical stimulation was significantly reduced only by APV+CNQX although APV or CNQX alone did not affect the expression, while c-Fos expression was reduced by APV and APV+CNQX but not by CNQX alone. These findings suggest that NMDA and AMPA/KA receptors are differentially involved in c-Fos and Zif/268 expression in the spinal dorsal horn following noxious thermal, formalin and mechanical stimulation.     

炎症反应通过上调CD36表达促进小鼠肾脏损伤

目的:探讨慢性炎症对小鼠肾脏CD36表达的影响及其在小鼠肾脏损伤中的作用。方法:将8周龄雄性C57BL/6J小鼠和CD36基因敲除(CD36KO)小鼠随机分为C57BL/6J生理盐水注射组、C57BL/6J酪蛋白注射组和CD36KO酪蛋白注射组,每组8只。高脂喂养处理14周后,收集小鼠血清、24 h尿液和肾组织样本。ELISA试剂盒检测血清中肿瘤坏死因子α(TNF-α)含量,全自动生化仪测定血、尿肾功能指标,HE染色和Masson染色分析肾脏病理改变,real-time PCR和Western blot检测肾脏组织中CD36及炎症/趋化因子(MCP-1、IL-6和TNF-α)m RNA和蛋白的表达,试剂盒测定组织内过氧化氢含量,免疫组化染色测定肾组织Nrf2和TGF-β1的蛋白表达。结果:与生理盐水注射组相比,酪蛋白注射能增强C57BL/6J小鼠血清TNF-α含量和肾组织中TNF-α的蛋白表达(P 0. 05),提示酪蛋白注射能成功诱导小鼠全身和肾脏局部的慢性炎症。同时酪蛋白注射显著促进了小鼠肾组织的CD36和TGF-β1蛋白表达,引起肾小球硬化、蛋白尿和血清肌酐含量显著增加,组织过氧化氢含量明显增加,Nrf2含量和抗氧化能力明显降低(P 0. 05)。而酪蛋白处理的CD36基因敲除小鼠肾组织病理学改变不明显,血、尿肾功能指标和尿量较酪蛋白处理的C57BL/6J小鼠明显降低,且肾组织过氧化氢含量低于酪蛋白处理的C57BL/6J小鼠(P 0. 05)。结论:炎症应激通过促进小鼠肾组织CD36表达,促进氧化应激,导致小鼠肾损伤。     

Participation of calbindin-D28K in nociception: results from calbindin-D<Subscript>28K</Subscript> knockout mice

Since calbindin-D_28K (CB-D_28K)-positive neurons have been related to nociceptive sensory processing, we have hypothesized that altered CB-D_28K expression could alter nociceptive transmission. We have used +/+ and −/− knockout (KO) mice for CB-D_28k in different behavioral models of pain and sensory responses at the caudalis subdivision of the trigeminal spinal nucleus in order to understand how this protein may participate in nociception. Behavioral responses to formalin injection in the hind paw or at the whisker pad or in the hind paw glutamate or i.p. acetic acid tests showed an increase of the pain threshold in CB-D_28k −/− mice. KO mice showed a diminution of the inhibitory activity at Sp5C nucleus and a marked reduction of GABA content. Sp5C neurons from CB-D_28k −/− mice did not change their spontaneous activity or tactile response after formalin injection in the whisker pad. In contrast, Sp5C neurons increased their spontaneous firing rate and tactile response after formalin injection in their receptive field in CB-D_28k +/+ mice. The results of this study demonstrate the active role played by CB-D_28k in nociceptive sensory transmission. The lack of this calcium binding protein, associated to deficient GABAergic neurotransmission, translates into dysfunction of sensory processing of nociceptive stimuli.     

Sustained acceleration of colonic transit following chronic homotypic stress in oxytocin knockout mice

Acute restraint stress delays gastric emptying and accelerates colonic transit via central corticotropin releasing factor (CRF) in rats. In contrast, central oxytocin has anxiolytic effects and attenuates the hypothalamus–pituitary–adrenal (HPA) axis in response to stress. Our recent study showed that up regulated oxytocin expression attenuates hypothalamic CRF expression and restores impaired gastric motility following chronic homotypic stress in mice. We studied the effects of acute and chronic homotypic stress on colonic transit and hypothalamic CRF mRNA expression in wild type (WT) and oxytocin knockout (OXT-KO) mice. Colonic transit was measured following acute restraint stress or chronic homotypic stress (repeated restraint stress for 5 consecutive days). ⁵¹Cr was injected via a catheter into the proximal colon. Ninety minutes after restraint stress loading, the entire colon was removed. The geometric center (GC) was calculated to evaluate colonic transit. Expression of CRF mRNA in the supraoptic nucleus (SON) was measured by real time RT-PCR. Colonic transit was significantly accelerated following acute stress in WT (GC = 8.1 ± 0.8; n = 7) and OXT KO mice (GC =9.4 ± 0.3; n = 7). The accelerated colonic transit was significantly attenuated in WT mice (GC = 6.6 ± 0.5; n = 9) following chronic homotypic stress while it was still accelerated in OXT KO mice (GC = 9.3 ± 0.5; n = 8). The increase in CRF mRNA expression at the SON was much greater in OXT-KO mice, compared to WT mice following chronic homotypic stress. It is suggested that oxytocin plays a pivotal role in mediating the adaptation mechanism following chronic homotypic stress in mice.     

Role of Surfactant Protein D in Experimental Otitis Media

Surfactant protein D (SP-D) is a C-type collectin and plays an important role in innate immunity and homeostasis in the lung. This study studied SP-D role in the nontypeable Haemophilus influenzae (NTHi)-induced otitis media (OM) mouse model. Wild-type C57BL/6 (WT) and SP-D knockout (KO) mice were used in this study. Mice were injected in the middle ear (ME) with 5 μL of NTHi bacterial solution (3.5 × 10⁵ CFU/ear) or with the same volume of sterile saline (control). Mice were sacrificed at 3 time points, days 1, 3, and 7, after treatment. We found SP-D expression in the Eustachian tube (ET) and ME mucosa of WT mice but not in SP-D KO mice. After infection, SP-D KO mice showed more intense inflammatory changes evidenced by the increased mucosal thickness and inflammatory cell infiltration in the ME and ET compared to WT mice (p < 0.05). Increased bacterial colony-forming units and cytokine (IL-6 and IL-1β) levels in the ear washing fluid of infected SP-D KO mice were compared to infected WT mice. Molecular analysis revealed higher levels of NF-κB and NLRP3 activation in infected SP-D KO compared to WT mice (p < 0.05). In vitro studies demonstrated that SP-D significantly induced NTHi bacterial aggregation and enhanced bacterial phagocytosis by macrophages (p < 0.05). Furthermore, human ME epithelial cells showed a dose-dependent increased expression of NLRP3 and SP-D proteins after LPS treatment. We conclude that SP-D plays a critical role in innate immunity and disease resolution through enhancing host defense and regulating inflammatory NF-κB and NLRP3 activation in experimental OM mice.     

Novel method for transdiaphragmatic pressure measurements in mice

The diaphragm muscle (DIAm) is responsible for breathing and determines the ability to generate both ventilatory and non-ventilatory behaviors. Size limitations of the mouse make transdiaphragmatic pressure (Pdi) measurement using a dual balloon system untenable. Adult C57BL/6J mice (n = 8) and C57BL/6 × 129 (n = 9), underwent Pdi measurements using solid-state pressure catheters spanning the thoracic and abdominal surfaces of the DIAm. Measurements were conducted during eupnea, hypoxia (10% O₂)–hypercapnia (5% CO₂), chemical airway stimulation (i.e., sneezing), spontaneously occurring deep breaths, sustained tracheal occlusion, and bilateral phrenic nerve stimulation. There was a difference in the Pdi generated across the range of ventilatory and non-ventilatory behaviors (p = 0.001). No difference in Pdi across behaviors was evident between mouse strains (p = 0.161). This study establishes a novel method to determine Pdi across a range of DIAm behaviors in mice that may be useful in evaluating conditions associated with reduced ability to perform expulsive, non-ventilatory behaviors.