Pharmacogenetic inhibition of TrkB signaling in adult mice attenuates mechanical hypersensitivity and improves locomotor function after spinal cord injury.

Brain-derived neurotrophic factor (BDNF) signals through tropomyosin receptor kinase B (TrkB), to exert various types of plasticity. The exact involvement of BDNF and TrkB in neuropathic pain states after spinal cord injury (SCI) remains unresolved. This study utilized transgenic TrkBF616 mice to examine the effect of pharmacogenetic inhibition of TrkB signaling, induced by treatment with 1NM-PP1 (1NMP) in drinking water for 5 days, on formalin-induced inflammatory pain, pain hypersensitivity, and locomotor dysfunction after thoracic spinal contusion. We also examined TrkB, ERK1/2, and pERK1/2 expression in the lumbar spinal cord and trunk skin. The results showed that formalin-induced pain responses were robustly attenuated in 1NMP-treated mice. Weekly assessment of tactile sensitivity with the von Frey test showed that treatment with 1NMP immediately after SCI blocked the development of mechanical hypersensitivity up to 4 weeks post-SCI. Contrastingly, when treatment started 2 weeks after SCI, 1NMP reversibly and partially attenuated hind-paw hypersensitivity. Locomotor scores were significantly improved in the early-treated 1NMP mice compared to late-treated or vehicle-treated SCI mice. 1NMP treatment attenuated SCI-induced increases in TrkB and pERK1/2 levels in the lumbar cord but failed to exert similar effects in the trunk skin. These results suggest that early onset TrkB signaling after SCI contributes to maladaptive plasticity that leads to spinal pain hypersensitivity and impaired locomotor function.

C-low threshold mechanoreceptor activation becomes sufficient to trigger affective pain in spinal cord-injured mice in association with increased respiratory rates.

The mechanisms of neuropathic pain after spinal cord injury (SCI) are not fully understood. In addition to the plasticity that occurs within the injured spinal cord, peripheral processes, such as hyperactivity of primary nociceptors, are critical to the expression of pain after SCI. In adult rats, truncal stimulation within the tuning range of C-low threshold mechanoreceptors (C-LTMRs) contributes to pain hypersensitivity and elevates respiratory rates (RRs) after SCI. This suggests that C-LTMRs, which normally encode pleasant, affiliative touch, undergo plasticity to transmit pain sensation following injury. Because tyrosine hydroxylase (TH) expression is a specific marker of C-LTMRs, in the periphery, here we used TH-Cre adult mice to investigate more specifically the involvement of C-LTMRs in at-level pain after thoracic contusion SCI. Using a modified light-dark chamber conditioned place aversion (CPA) paradigm, we assessed chamber preferences and transitions between chambers at baseline, and in response to mechanical and optogenetic stimulation of C-LTMRs. In parallel, at baseline and select post-surgical timepoints, mice underwent non-contact RR recordings and von Frey assessment of mechanical hypersensitivity. The results showed that SCI mice avoided the chamber associated with C-LTMR stimulation, an effect that was more pronounced with optical stimulation. They also displayed elevated RRs at rest and during CPA training sessions. Importantly, these changes were restricted to chronic post-surgery timepoints, when hindpaw mechanical hypersensitivity was also evident. Together, these results suggest that C-LTMR afferent plasticity, coexisting with potentially facilitatory changes in breathing, drives at-level affective pain following SCI in adult mice.

Sodium bicarbonate cotransporter NBCn1/Slc4a7 affects locomotor activity and hearing in mice.

Despite a widespread expression pattern in the central nervous system, the role of the sodium bicarbonate cotransporter NBCn1/Slc4a7 has not been investigated for locomotor activity, emotion and cognition. Here, we addressed the behavioral consequences of NBCn1 knockout and evaluated hearing and vision that are reportedly impaired in an earlier line of NBCn1 knockout mice and may contribute to behavioral changes. In a circular open field, the knockout mice traveled a shorter distance, especially in the periphery of the chamber, than wildtype littermates. The knockout mice also traveled a shorter total distance in a home cage-like open field. Rearing and grooming behaviors were reduced. The knockout and control mice displayed similar time spent and number of open and closed arms in the elevated plus maze test, indicating negligible change in anxiety. In the Morris water maze test, both groups of mice learned the location of an escape platform within comparable time on the training trials and showed similar platform identification on the probe trial. The knockout mice maintained normal visual responses in the optokinetic drum and produced evoked potentials in response to light stimuli. However, these mice failed to produce auditory evoked potentials. qPCR revealed a robust expression of an alternatively transcribed NBCn1 variant in the knockout mouse retina. These results indicate that NBCn1 deletion leads to reduced locomotor activity in mice by affecting their exploratory behaviors or emotionality. The deletion also causes hearing loss, but its effect on vision varies between different lines of knockout mice.

Elderly woman with subacute lower limb weakness and rapid systemic decline.

A 74-year-old woman developed bilateral leg weakness, with fluctuating cognitive and systemic symptoms that progressed despite treatment. Her diagnosis was confirmed at autopsy. Her case was discussed at the Edinburgh Clinical Neurology Course 2019 Clinicopathological Conference.

Hypothesis: Pulmonary Afferent Activity Patterns During Slow, Deep Breathing Contribute to the Neural Induction of Physiological Relaxation.

Control of respiration provides a powerful voluntary portal to entrain and modulate central autonomic networks. Slowing and deepening breathing as a relaxation technique has shown promise in a variety of cardiorespiratory and stress-related disorders, but few studies have investigated the physiological mechanisms conferring its benefits. Recent evidence suggests that breathing at a frequency near 0.1 Hz (6 breaths per minute) promotes behavioral relaxation and baroreflex resonance effects that maximize heart rate variability. Breathing around this frequency appears to elicit resonant and coherent features in neuro-mechanical interactions that optimize physiological function. Here we explore the neurophysiology of slow, deep breathing and propose that coincident features of respiratory and baroreceptor afferent activity cycling at 0.1 Hz entrain central autonomic networks. An important role is assigned to the preferential recruitment of slowly-adapting pulmonary afferents (SARs) during prolonged inhalations. These afferents project to discrete areas in the brainstem within the nucleus of the solitary tract (NTS) and initiate inhibitory actions on downstream targets. Conversely, deep exhalations terminate SAR activity and activate arterial baroreceptors via increases in blood pressure to stimulate, through NTS projections, parasympathetic outflow to the heart. Reciprocal SAR and baroreceptor afferent-evoked actions combine to enhance sympathetic activity during inhalation and parasympathetic activity during exhalation, respectively. This leads to pronounced heart rate variability in phase with the respiratory cycle (respiratory sinus arrhythmia) and improved ventilation-perfusion matching. NTS relay neurons project extensively to areas of the central autonomic network to encode important features of the breathing pattern that may modulate anxiety, arousal, and attention. In our model, pronounced respiratory rhythms during slow, deep breathing also support expression of slow cortical rhythms to induce a functional state of alert relaxation, and, via nasal respiration-based actions on olfactory signaling, recruit hippocampal pathways to boost memory consolidation. Collectively, we assert that the neurophysiological processes recruited during slow, deep breathing enhance the cognitive and behavioral therapeutic outcomes obtained through various mind-body practices. Future studies are required to better understand the physio-behavioral processes involved, including in animal models that control for confounding factors such as expectancy biases.

Spontaneous and Stimulus-Evoked Respiratory Rate Elevation Corresponds to Development of Allodynia in Spinal Cord-Injured Rats.

Respiratory complications frequently accompany spinal cord injury (SCI) and slowed breathing has been shown to mitigate pain sensitivity. It is possible that elevated respiratory rates (RRs) signal the emergence of chronic pain after SCI. We previously validated the use of remote electric field sensors to noninvasively track breathing in freely behaving rodents. Here, we examined spontaneous (resting) and stimulus-evoked RRs as potential indices of mechanical hypersensitivity following SCI. Adult male Long-Evans rats received a lower thoracic hemisection or contusion SCI, or sham surgery, and underwent weekly assessments of mechanical and thermal sensitivity using the von Frey and Hargreaves tests, respectively. Resting RRs were recorded with remote sensors prior to nociception assays as well as 1 day post-surgery. Evoked RRs were quantified weekly in response to at-level mechanical stimulation provided by a small brush at various stimulation speeds, including those corresponding to the distinct tuning properties of a sub-population of cutaneous afferents known as C-low threshold mechanoreceptors. SCI rats developed mechanical hypersensitivity, which peaked 2-3 weeks after SCI. Compared with at baseline, hemisection SCI rats showed significantly heightened resting RRs at 1 day and 7 days post-injury, and the latter predicted development of pain hypersensitivity. In contusion SCI rats, resting RR increases were less substantial but occurred at all weekly time-points. Increases in brush-evoked RR coincided with full expression of hypersensitivity at 14 (hemisection) or 21 (contusion) days after SCI, and these effects were restricted to the lowest brush speeds. Our results support the possibility that early changes in RR may convey pain information in rats.

Slow Breathing Can Be Operantly Conditioned in the Rat and May Reduce Sensitivity to Experimental Stressors.

In humans, exercises involving slowed respiratory rate (SRR) counter autonomic sympathetic bias and reduce responses to stressors, including in individuals with various degrees of autonomic dysfunction. In the rat, we examined whether operant conditioning could lead to reductions in respiratory rate (RR) and performed preliminary studies to assess whether conditioned SRR was sufficient to decrease physiological and behavioral responsiveness to stressors. RR was continuously monitored during 20 2-h sessions using whole body plethysmography. SRR conditioned, but not yoked control rats, were able to turn off aversive visual stimulation (intermittent bright light) by slowing their breathing below a preset target of 80 breaths/min. SRR conditioned rats greatly increased the incidence of breaths below the target RR over training, with average resting RR decreasing from 92 to 81 breaths/min. These effects were significant as a group and vs. yoked controls. Preliminary studies in a subset of conditioned rats revealed behavioral changes suggestive of reduced reactivity to stressful and nociceptive stimuli. In these same rats, intermittent sessions without visual reinforcement and a post-training priming stressor (acute restraint) demonstrated that conditioned rats retained reduced RR vs. controls in the absence of conditioning. In conclusion, we present the first successful attempt to operantly condition reduced RR in an animal model. Although further studies are needed to clarify the physio-behavioral concomitants of slowed breathing, the developed model may aid subsequent neurophysiological inquiries on the role of slow breathing in stress reduction.

Use of electric field sensors for recording respiration, heart rate, and stereotyped motor behaviors in the rodent home cage.

BACKGROUND: Numerous environmental and genetic factors can contribute significantly to behavioral and cardiorespiratory variability observed experimentally. Affordable technologies that allow for noninvasive home cage capture of physio-behavioral variables should enhance understanding of inter-animal variability including after experimental interventions. NEW METHOD: We assessed whether EPIC electric field sensors (Plessey Semiconductors) embedded within or attached externally to a rodent's home cage could accurately record respiration, heart rate, and motor behaviors. COMPARISON WITH EXISTING METHODS: Current systems for quantification of behavioral variables require expensive specialty equipment, while measures of respiratory and heart rate are often provided by surgically implanted or chronically affixed devices. RESULTS: Sensors accurately encoded imposed sinusoidal changes in electric field tested at frequencies ranging from 0.5-100Hz. Mini-metronome arm movements were easily detected, but response magnitude was highly distance dependent. Sensors accurately reported respiration during whole-body plethysmography. In anesthetized rodents, PVC tube-embedded sensors provided accurate mechanical detection of both respiratory and heart rate. Comparable success was seen in naturally behaving animals at rest or sleeping when sensors were attached externally. Video-verified motor behaviors (sniffing, grooming, chewing, and rearing) were detectable and largely separable by their characteristic voltage fluctuations. Larger movement-related events had comparably larger voltage dynamics that easily allowed for a broad approximation of overall motor activity. Spectrograms were used to quickly depict characteristic frequencies in long-lasting recordings, while filtering and thresholding software allowed for detection and quantification of movement-related physio-behavioral events. CONCLUSIONS: EPIC electric field sensors provide a means for affordable non-contact home cage detection of physio-behavioral variables.

Pulmonary Mycobacterium marinum infection: 'fish tank granuloma' of the lung.

A 65-year-old man presented with a six-month history of lethargy, weight loss and dry cough. He had a background of mild chronic obstructive pulmonary disease. Chest radiograph showed new right upper lobe cavitary opacification. Sputum cultures were acid-fast bacilli smear positive and yielded Mycobacterium marinum - a non-tuberculous mycobacterium (NTM) often found in aquatic environments and rarely associated with respiratory disease. The suspected source was silent aspiration of contaminated water, likely due to his initiating the siphon of his fish-tank by mouth. He completed a one-year course of rifampicin, ethambutol and clarithromycin, with negative repeat sputum mycobacteria cultures and radiological improvement. This case report demonstrates a successful approach to investigation and further management of Mycobacterium marinum pulmonary disease - a rare condition, particularly in immunocompetent individuals, with limited treatment guidelines.

Sputum trace metals are biomarkers of inflammatory and suppurative lung disease.

BACKGROUND: Induced sputum cytology and protein biomarkers can be used to assess airways inflammation. Increases in sputum iron have been described in inflammatory lung disease. We hypothesized that other sputum metals may be affected by airways inflammation and investigated their potential value as biomarkers. METHODS: Sputum was obtained from 20 healthy control subjects and from patients with inflammatory pulmonary diseases (23 with cystic fibrosis [CF], 16 with bronchiectasis, 17 with asthma, and 23 with COPD), and iron, zinc, manganese, and copper were measured. Fourteen patients with CF were also studied through an exacerbation cycle. RESULTS: Sputum zinc and iron were elevated in CF and non-CF bronchiectasis vs controls (P < .001, zinc; P < .01 iron). Manganese was elevated in asthma (P < .01) and bronchiectasis (P < .05) vs controls. Copper was elevated in CF vs controls (P < .05). Zinc decreased (P < .01) following treatment of CF exacerbation. In subjects with CF zinc levels correlated with other biomarkers. CONCLUSIONS: These results suggest a relationship of high concentrations of total zinc and iron with airways inflammation in CF and non-CF bronchiectasis, with longitudinal changes being observed in CF. Further work is required to elucidate potential inflammatory mechanisms related to these observations.

Sputum proteomics in inflammatory and suppurative respiratory diseases.

RATIONALE: Markers of inflammatory activity are important for assessment and management of many respiratory diseases. Markers that are currently unrecognized may be more valuable than those presently believed to be useful. OBJECTIVES: To identify potential biomarkers of suppurative and inflammatory lung disease in induced sputum samples. METHODS: Induced sputum was collected from 20 healthy control subjects, 24 patients with asthma, 24 with chronic obstructive pulmonary disease, 28 with cystic fibrosis (CF), and 19 with bronchiectasis. Twelve patients with CF had sputum sampled before and after antibiotic therapy for an infective exacerbation. The fluid phase of induced sputum was analyzed by surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectroscopy on three protein array surfaces. Some protein markers were selected for identification, and relevant ELISA assays sought. For 12 patients with CF, both SELDI-TOF and ELISA monitored changes in inflammatory responses during infective exacerbations. MEASUREMENTS AND MAIN RESULTS: SELDI-TOF identified potential biomarkers that differentiated each of the disease groups from healthy control subjects: at a significance of P < 0.01, there were 105 for asthma, 113 for chronic obstructive pulmonary disease, 381 for CF, and 377 for bronchiectasis. Peaks selected for protein identification yielded calgranulin A, calgranulin B, calgranulin C, Clara cell secretory protein, lysosyme c, proline rich salivary peptide, cystatin s, and hemoglobin alpha. On treatment of an infective CF exacerbation, SELDI-TOF determined falls in levels of calgranulin A and calgranulin B that were mirrored by ELISA-measured falls in calprotectin (heterodimer of calgranulins A and B). CONCLUSIONS: Proteomic screening of sputum yields potential biomarkers of inflammation. The early development of a clinically relevant assay from such data is demonstrated.

A genome-wide screen identifies a single beta-defensin gene cluster in the chicken: implications for the origin and evolution of mammalian defensins.

BACKGROUND: Defensins comprise a large family of cationic antimicrobial peptides that are characterized by the presence of a conserved cysteine-rich defensin motif. Based on the spacing pattern of cysteines, these defensins are broadly divided into five groups, namely plant, invertebrate, alpha-, beta-, and theta-defensins, with the last three groups being mostly found in mammalian species. However, the evolutionary relationships among these five groups of defensins remain controversial. RESULTS: Following a comprehensive screen, here we report that the chicken genome encodes a total of 13 different beta-defensins but with no other groups of defensins being discovered. These chicken beta-defensin genes, designated as Gallinacin 1-13, are clustered densely within a 86-Kb distance on the chromosome 3q3.5-q3.7. The deduced peptides vary from 63 to 104 amino acid residues in length sharing the characteristic defensin motif. Based on the tissue expression pattern, 13 beta-defensin genes can be divided into two subgroups with Gallinacin 1-7 being predominantly expressed in bone marrow and the respiratory tract and the remaining genes being restricted to liver and the urogenital tract. Comparative analysis of the defensin clusters among chicken, mouse, and human suggested that vertebrate defensins have evolved from a single beta-defensin-like gene, which has undergone rapid duplication, diversification, and translocation in various vertebrate lineages during evolution. CONCLUSIONS: We conclude that the chicken genome encodes only beta-defensin sequences and that all mammalian defensins are evolved from a common beta-defensin-like ancestor. The alpha-defensins arose from beta-defensins by gene duplication, which may have occurred after the divergence of mammals from other vertebrates, and theta-defensins have arisen from alpha-defensins specific to the primate lineage. Further analysis of these defensins in different vertebrate lineages will shed light on the mechanisms of host defense and evolution of innate immunity.