Rat Diaphragm Research Articles

Potentially zoonotic pathogens and parasites in opportunistically sourced urban brown rats (Rattus norvegicus) in and around Helsinki, Finland, 2018 to 2023.

BackgroundBrown rats (Rattus norvegicus) are synanthropic rodents with worldwide distribution, which are known to harbour many zoonotic pathogens and parasites. No systematic zoonotic surveys targeting multiple pathogens and parasites have previously been conducted in urban rats in Finland.AimIn Helsinki, Finland, we explored the presence and prevalence in brown rats of certain pathogens and parasites (including helminths, viruses and bacteria) across potentially zoonotic taxa.MethodsWe opportunistically received rat carcasses from pest management operators and citizens from 2018 to 2023. We searched for heart- or lungworms, performed rat diaphragm digestion to check for Trichinella and morphologically identified intestinal helminths. We assessed virus exposure by immunofluorescence assay or PCR, and detected bacteria by PCR (Leptospira) or culture (Campylobacter).ResultsAmong the rats investigated for helminths, no heart- or lungworms or Trichinella species were detected and the most common finding was the cestode Hymenolepis nana (in 9.7% of individuals sampled, 28/288). For some of the surveyed virus taxa, several rats were seropositive (orthopoxviruses, 5.2%, 11/211; arenaviruses, 2.8%, 6/211; hantaviruses 5.2%, 11/211) or tested positive by PCR (rat hepatitis E virus, 1.8%, 4/216). Campylobacter jejuni (6.6%, 17/259) and Leptospira interrogans (1.2%, 2/163) bacteria were also present in the rat population examined.ConclusionsPrevalences of potentially zoonotic pathogens and parasites in brown rats in Helsinki appeared low. This may explain low or non-existent diagnosis levels of rat-borne pathogen and parasite infections reported in people there. Nevertheless, further assessment of under-diagnosis, which cannot be excluded, would enhance understanding the risks of zoonoses.

Changes in contractile characteristics of rat skeletal muscles associated with P2-receptor activation after spinal cord transection

Introduction. Traumatic spinal cord and peripheral-nerve injury is associated with release of proinflammatory cytokines and chemokines, which may stimulate neuronal activity. Adenosine triphosphoric acid (ATP) is an important pain mediator involved in the acute and chronic neuropathic pain development. Its excessive release from primary injured tissue leads to activation of P2-receptors, which may further start secondary injury mechanisms. Although the effects of ATP on the peripheral nervous system are relatively well studied, the pathophysiological role of purinergic signaling after spinalization remains unclear. The study was aimed at assessing the post-spinalization effects of P2-receptors on the contractile characteristics of rat skeleton muscles. Materials and methods. The objects of the study were the soleus muscle, the extensor digitorum longus (EDL) muscle, and diaphragm in intact rats and spinalized rats. Seven days after laminectomy followed by spinal cord transection, animals were anesthetized, exsanguinated, and their muscles with nerve stumps were isolated. Contractile response parameters were recorded using mechanomyography (MMG). To study effects of ATP on ligand binding, ATP was added to a bath and mechanical responses in the rat muscles were assessed 7 min after. After washing with Krebs–Henseleit solution, the preparations were incubated with suramin solution for 20 min with subsequent ATP application. Then the mechanical responses in the muscles were again recorded. Statistical significance was assessed using Student's t-test for independent (unpaired) and paired samples. Results. We found a significant (p 0.05) decrease in the modulating activity of ATP, as the main endogenous signaling agent, in the cholinergic synapse of the soleus muscle from 32.4 to 5.8% and from 13.7 to 5.6% for the EDL muscle after the spinalization (spinal cord injury at the Th6–Th7 level) compared with intact animals. No such dramatic changes were observed in the diaphragm. Conclusions. Abnormal ATP-mediated modulation of neuromuscular transmission demonstrated in this study supports the involvement of purinergic signaling in the neurotrophic control and functioning of various motor units.

Synthesis and Biological Evaluation of Novel MB327 Analogs as Resensitizers for Desensitized Nicotinic Acetylcholine Receptors after Intoxication with Nerve Agents

Poisoning with organophosphorus compounds, which can lead to a cholinergic crisis due to the inhibition of acetylcholinesterase and the subsequent accumulation of acetylcholine (ACh) in the synaptic cleft, is a serious problem for which treatment options are currently insufficient. Our approach to broadening the therapeutic spectrum is to use agents that interact directly with desensitized nicotinic acetylcholine receptors (nAChRs) in order to induce functional recovery after ACh overstimulation. Although MB327, one of the most prominent compounds investigated in this context, has already shown positive properties in terms of muscle force recovery, this compound is not suitable for use as a therapeutic agent due to its insufficient potency. By means of in silico studies based on our recently presented allosteric binding pocket at the nAChR, i.e. the MB327-PAM-1 binding site, three promising MB327 analogs with a 4-aminopyridinium ion partial structure (PTM0056, PTM0062 and PTM0063) were identified. In this study, we present the synthesis and biological evaluation of a series of new analogs of the aforementioned compounds with a 4-aminopyridinium ion partial structure (PTM0064-PTM0072), as well as hydroxy-substituted analogs of MB327 (PTMD90-0012 and PTMD90-0015) designed to substitute entropically unfavorable water clusters identified during molecular dynamics simulations. The compounds were characterized in terms of their binding affinity towards the aforementioned binding site by applying the UNC0642 MS Binding Assays and in terms of their muscle force reactivation in rat diaphragm myography. More potent compounds were identified compared to MB327, as some of them showed a higher affinity towards MB327-PAM-1 and also a higher recovery of neuromuscular transmission at lower compound concentrations. To improve the treatment of organophosphate poisoning, direct targeting of nAChRs with appropriate compounds is a key step, and this study is an important contribution to this research.

Impact of Reduced Activity on Endurance of Rat Diaphragm Muscle

The diaphragm muscle (DIAm) as the major inspiratory pump is highly active. During breathing, fatigue resistant DIAm motor units are recruited that comprise type I and IIa fibers. Accordingly, these fibers have substantially higher mitochondrial volume density (MVD) and mitochondrial respiratory capacity (SDHmax) compared to more fatigable type IIx/IIb fibers. Cervical spinal cord injury disrupts descending inspiratory drive and markedly reduces eupneic activity of the DIAm. With C2 spinal cord hemisection (C2SH) DIAm activity on the ipsilateral side is reduced. Following a two-week period of C2SH, we found reduced MVD and SDHmax in type I and IIa DIAm fibers. As a result, we hypothesize that endurance of the DIAm is reduced. Female and male Sprague Dawley rats were used. EMG electrodes were placed in the left and right DIAm 3 days prior to C2SH surgery, and baseline recordings of eupneic DIAm activity were obtained in awake animals. The right C2 spinal cord was hemisected, and the extent of injury was confirmed by the absence of eupneic DIAm activity on the ipsilateral side while animals were anesthetized. Subsequently, DIAm EMG activity was recorded at 3, 7 and 14 days post C2SH. After two weeks, rats were anesthetized and the DIAm was excised. DIAm strips were cut and suspended in a tissue chamber containing Rees-Simpson solution maintained at 26oC, with thecostal margin clamped and the central tendon attached to a Cambridge Dual-Mode force/length transducer. DIAm strips were stimulated using platinum plate electrodes with supramaximal current (~250 mA) pulses (1 ms), and maximum specific force was determined at 75 Hz. The force-velocity relationship was then determined by systematically changing load, and maximum velocity (Vmax) was estimated based on curve fitting with the Hill-equation. Based on the force-velocity measurements peak power of the DIAm was calculated. Subsequently, the DIAm was repetitively stimulated and allowed to shorten at 30% maximum velocity (isovelocity approximating peak power output). Endurance of the DIAm was then determined as the duration that peak power output was sustained. Finally, the DIAm was rapidly frozen, and cross-sections were cut for fiber type and cross-sectional area (CSA) measurements. C2SH resulted in a marked decrease in eupneic DIAm EMG activity across the two-week period, which disproportionately affected type I and IIa DIAm fibers. However, there was no impact of C2SH on DIAm fiber type proportions or CSA. Following two-weeks C2SH, maximum specific force, Vmax, and peak power output of the DIAm were comparable between intact and injured sides. However, endurance of the DIAm was reduced on the injured side compared to the intact side. These results support our hypothesis that reduced activity of DIAm, reduces oxidative capacity of type I and IIa fibers and thereby reduces endurance. In clinical conditions such as mechanical ventilation DIAm activity is reduced and if maintained for longer periods of time this may affect DIAm endurance post weaning. Research supported by NIH grant: HL146114 (GCS). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Assessing Rat Diaphragm Motor Unit Connectivity Outcome Measures as Quantitative Biomarkers of Phrenic Motor Neuron Degeneration and Compensation.

Loss of ventilatory muscle function is a consequence of motor neuron injury and neurodegeneration (e.g., cervical spinal cord injury and amyotrophic lateral sclerosis, respectively). Phrenic motor neurons are the final link between the central nervous system and muscle, and their respective motor units (groups of muscle fibers innervated by a single motor neuron) represent the smallest functional unit of the neuromuscular ventilatory system. Compound muscle action potential (CMAP), single motor unit potential (SMUP), and motor unit number estimation (MUNE) are established electrophysiological approaches that enable the longitudinal assessment of motor unit integrity in animal models over time but have mostly been applied to limb muscles. Therefore, the objectives of this study are to describe an approach in preclinical rodent studies that can be used longitudinally to quantify the phrenic MUNE, motor unit size (represented as SMUP), and CMAP, and then to demonstrate the utility of these approaches in a motor neuron loss model. Sensitive, objective, and translationally relevant biomarkers for neuronal injury, degeneration, and regeneration in motor neuron injury and diseases can significantly aid and accelerate experimental research discoveries to clinical testing.

Restoration of nerve agent impaired neuromuscular transmission in rat diaphragm by bispyridinium non-oximes – Structure-activity relationships

Organophosphate (OP) poisoning is currently treated with atropine, oximes and benzodiazepines. The nicotinic signs, i.e., respiratory impairment, can only be targeted indirectly via the use of oximes as reactivators of OP-inhibited acetylcholinesterase. Hence, compounds selectively targeting nicotinic acetylcholine receptors (nAChRs) might fundamentally improve current treatment options. The bispyridinium compound MB327 has previously shown some therapeutic effect against nerve agents in vitro and in vivo. Nevertheless, compound optimization was deemed necessary, due to limitations (e.g., toxicity and efficacy). The current study investigated a series of 4-tert-butyl bispyridinium compounds and of corresponding bispyridinium compounds without substituents in a rat diaphragm model using an indirect field stimulation technique. The length of the respective linker influenced the ability of the bispyridinium compounds to restore muscle function in rat hemidiaphragms. The current data show structure-activity relationships for a series of bispyridinium compounds and provide insight for future structure-based molecular modeling.

Pharmacological effects of monoterpene carveol on the neuromuscular system of nematodes and mammals.

The control of parasitic nematode infections relies mostly on anthelmintics. The potential pharmacotherapeutic application of phytochemicals, in order to overcome parasite resistance and enhance the effect of existing drugs, is becoming increasingly important. The antinematodal effects of carveol was tested on the free-living nematode Caenorhabditis elegans and the neuromuscular preparation of the parasitic nematode Ascaris suum. Carveol caused spastic paralysis in C. elegans. In A. suum carveol potentiated contractions induced by acetylcholine (ACh) and this effect was confirmed with two-electrode voltage-clamp electrophysiology on the A. suum nicotinic ACh receptor expressed in Xenopus oocytes. However, potentiating effect of carveol on ACh-induced contractions was partially sensitive to atropine, indicates a dominant nicotine effect but also the involvement of some muscarinic structures. The effects of carveol on the neuromuscular system of mammals are also specific. In micromolar concentrations, carveol acts as a non-competitive ACh antagonist on ileum contractions. Unlike atropine, it does not change the EC50 of ACh, but reduces the amplitude of contractions. Carveol caused an increase in Electrical Field Stimulation-evoked contractions of the isolated rat diaphragm, but at higher concentrations it caused an inhibition. Also, carveol neutralized the mecamylamine-induced tetanic fade, indicating a possibly different pre- and post-synaptic action at the neuromuscular junction.

MS Binding Assays with UNC0642 as reporter ligand for the MB327 binding site of the nicotinic acetylcholine receptor

Intoxications with organophosphorus compounds (OPCs) based chemical warfare agents and insecticides may result in a detrimental overstimulation of muscarinic and nicotinic acetylcholine receptors evolving into a cholinergic crisis leading to death due to respiratory failure. In the case of the nicotinic acetylcholine receptor (nAChR), overstimulation leads to a desensitization of the receptor, which cannot be pharmacologically treated so far. Still, compounds interacting with the MB327 binding site of the nAChR like the bispyridinium salt MB327 have been found to re-establish the functional activity of the desensitized receptor. Only recently, a series of quinazoline derivatives with UNC0642 as one of the most prominent representatives has been identified to address the MB327 binding site of the nAChR, as well. In this study, UNC0642 has been utilized as a reporter ligand to establish new Binding Assays for this target. These assays follow the concept of MS Binding Assays for which by assessing the amount of bound reporter ligand by mass spectrometry no radiolabeled material is required. According to the results of the performed MS Binding Assays comprising saturation and competition experiments it can be concluded, that UNC0642 used as a reporter ligand addresses the MB327 binding site of the Torpedo-nAChR. This is further supported by the outcome of ex vivo studies carried out with poisoned rat diaphragm muscles as well as by in silico studies predicting the binding mode of UNC0646, an analog of UNC0642 with the highest binding affinity, in the recently proposed binding site of MB327 (MB327-PAM-1). With UNC0642 addressing the MB327 binding site of the Torpedo-nAChR, this and related quinazoline derivatives represent a promising starting point for the development of novel ligands of the nAChR as antidotes for the treatment of intoxications with organophosphorus compounds. Further, the new MS Binding Assays are a potent alternative to established assays and of particular value, as they do not require the use of radiolabeled material and are based on a commercially available compound as reporter ligand, UNC0642, exhibiting one of the highest binding affinities for the MB327 binding site known so far.

Pulmonary hypertension alters blood flow distribution and impairs the hyperemic response in the rat diaphragm.

Pulmonary hypertension (PH) is characterized by pulmonary vascular remodeling, respiratory muscle and cardiac impairments, and exercise intolerance. Specifically, impaired gas exchange increases work of the diaphragm; however, compromised contractile function precludes the diaphragm from meeting the increased metabolic demand of chronic hyperventilation in PH. Given that muscle contractile function is in part, dependent upon adequate blood flow (), diaphragmatic dysfunction may be predicated by an inability to match oxygen delivery with oxygen demand. We hypothesized that PH rats would demonstrate a decreased hyperemic response to contractions compared to healthy controls. Methods: Sprague-Dawley rats were randomized into healthy (HC, n = 7) or PH (n = 7) groups. PH rats were administered monocrotaline (MCT) while HC rats received vehicle. Disease progression was monitored via echocardiography. Regional and total diaphragm blood flow and vascular conductance at baseline and during 3min of electrically-stimulated contractions were determined using fluorescent microspheres. Results: PH rats displayed morphometric and echocardiographic criteria for disease (i.e., acceleration time/ejection time, right ventricular hypertrophy). In all rats, total costal diaphragm increased during contractions and did not differ between groups. In HC rats, there was a greater increase in medial costal compared to PH rats (55% ± 3% vs. 44% ± 4%, p < 0.05), who demonstrated a redistribution of to the ventral costal region. Conclusion: These findings support a redistribution of regional diaphragm perfusion and an impaired medial costal hyperemic response in PH, suggesting that PH alters diaphragm vascular function and oxygen delivery, providing a potential mechanism for PH-induced diaphragm contractile dysfunction.

Age-related gene expression signatures from limb skeletal muscles and the diaphragm in mice and rats reveal common and species-specific changes

BackgroundAs a result of aging, skeletal muscle undergoes atrophy and a decrease in function. This age-related skeletal muscle weakness is known as “sarcopenia”. Sarcopenia is part of the frailty observed in humans. In order to discover treatments for sarcopenia, it is necessary to determine appropriate preclinical models and the genes and signaling pathways that change with age in these models.Methods and resultsTo understand the changes in gene expression that occur as a result of aging in skeletal muscles, we generated a multi-time-point gene expression signature throughout the lifespan of mice and rats, as these are the most commonly used species in preclinical research and intervention testing. Gastrocnemius, tibialis anterior, soleus, and diaphragm muscles from male and female C57Bl/6J mice and male Sprague Dawley rats were analyzed at ages 6, 12, 18, 21, 24, and 27 months, plus an additional 9-month group was used for rats. More age-related genes were identified in rat skeletal muscles compared with mice; this was consistent with the finding that rat muscles undergo more robust age-related decline in mass. In both species, pathways associated with innate immunity and inflammation linearly increased with age. Pathways linked with extracellular matrix remodeling were also universally downregulated. Interestingly, late downregulated pathways were exclusively found in the rat limb muscles and these were linked to metabolism and mitochondrial respiration; this was not seen in the mouse.ConclusionsThis extensive, side-by-side transcriptomic profiling shows that the skeletal muscle in rats is impacted more by aging compared with mice, and the pattern of decline in the rat may be more representative of the human. The observed changes point to potential therapeutic interventions to avoid age-related decline in skeletal muscle function.

Development and systematic evaluation of decellularization protocols in different application models for diaphragmatic tissue engineering

BackgroundTissue engineered bioscaffolds based on decellularized composites have gained increasing interest for treatment of various diaphragmatic impairments, including muscular atrophies and diaphragmatic hernias. Detergent-enzymatic treatment (DET) constitutes a standard strategy for diaphragmatic decellularization. However, there is scarce data on comparing DET protocols with different substances in distinct application models in their ability to maximize cellular removal while minimizing extracellular matrix (ECM) damage. MethodsWe decellularized diaphragms of male Sprague Dawley rats with 1 % or 0.1 % sodium dodecyl sulfate (SDS) and 4 % sodium deoxycholate (SDC) by orbital shaking (OS) or retrograde perfusion (RP) through the vena cava. We evaluated decellularized diaphragmatic samples by (1) quantitative analysis including DNA quantification and biomechanical testing, (2) qualitative and semiquantitative analysis by proteomics, as well as (3) qualitative assessment with macroscopic and microscopic evaluation by histological staining, immunohistochemistry and scanning electron microscopy. ResultsAll protocols produced decellularized matrices with micro- and ultramorphologically intact architecture and adequate biomechanical performance with gradual differences. The proteomic profile of decellularized matrices contained a broad range of primal core and ECM-associated proteins similar to native muscle. While no outstanding preference for one singular protocol was determinable, SDS-treated samples showed slightly beneficial properties in comparison to SDC-processed counterparts. Both application modalities proved suitable for DET. ConclusionDET with SDS or SDC via orbital shaking or retrograde perfusion constitute suitable methods to produce adequately decellularized matrices with characteristically preserved proteomic composition. Exposing compositional and functional specifics of variously treated grafts may enable establishing an ideal processing strategy to sustain valuable tissue characteristics and optimize consecutive recellularization. This aims to design an optimal bioscaffold for future transplantation in quantitative and qualitative diaphragmatic defects.

Effect of aging on maximum shortening velocity, peak power output and endurance of rat diaphragm muscle

Sarcopenia is defined as age-related atrophy of muscle fibers as well as a decrease in specific force (force per cross-sectional area). In the rat diaphragm muscle (DIAm), sarcopenia primarily affects type IIx/IIb fibers that generate greater specific force, have the fastest maximum shortening velocities, but display greater fatigue with repeated activation. We hypothesized that as a result of sarcopenia, maximum shortening velocity and power output of the DIAm decrease with age. DIAm strips ~2-3 mm wide were excised from 6-month and 24-month female (n=4 per age group) and male (n=4 per age group) rats. DIAm strips were suspended in a tissue chamber containing Rees–Simpson solution maintained at 26oC, with the costal margin clamped and the central tendon attached to a Cambridge Dual-Mode force/length transducer. To determine maximum isometric specific force, DIAm strips were stimulated using platinum plate electrodes with supramaximal current (~250 mA) pulses (1 ms) at 75 Hz delivered in 1-s duration train. The muscle strips were then allowed to shorten over a 30-ms period against different percentages of maximum isometric load. Based on the load/velocity relationship, the maximum shortening velocity (Vmax) and peak power output were determined. Subsequently, the muscle strip was repetitively stimulated and allowed to shorten at 30% maximum load (approximating peak power output). In this protocol, the muscle strip was stimulated at 75 Hz an generated isometric force during the initial 225 ms of the 330 ms duration train the muscle. Thereafter, the load was reduced to 30% maximum and the muscle strip was allowed to shorten for the subsequent 105 ms. Finally, the DIAm strip was stretched to ~150% its resting length (corresponding to optimal sarcomere length) and rapidly frozen in melting isopentane. Transverse serial sections of DIAm fibers were cut, and fiber types identified by immunoreactivity to specific myosin heavy chain (MyHC) isoforms. As previously observed, the cross-sectional areas of type IIx/IIb DIAm fibers and maximum isometric specific force were reduced in 24-month compared to 6-month rats confirming sarcopenia. The maximum shortening velocity of the DIAm in 24-month rats was slower than in the 6-month rats and peak power output of the DIAm was reduced in 24-month rats. The duration during which peak power output could be maintained (i.e., endurance) of the DIAm was prolonged in 24-month compared to 6-month rats (120 s vs 60 s, respectively). These results support our hypothesis that aging affects maximum shortening velocity and peak power output of the DIAm. It is important to note that endurance of the DIAm is preserved, consistent with resilience of breathing motor function in old age. Research supported by NIH grant AG44615 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Association of respiratory failure with inhibition of NaV1.6 in the phrenic nerve

ABSTRACT As part of a drug discovery effort to identify potent inhibitors of NaV1.7 for the treatment of pain, we observed that inhibitors produced unexpected cardiovascular and respiratory effects in vivo. Specifically, inhibitors administered to rodents produced changes in cardiovascular parameters and respiratory cessation. We sought to determine the mechanism of the in vivo adverse effects by studying the selectivity of the compounds on NaV1.5, NaV1.4, and NaV1.6 in in vitro and ex vivo assays. Inhibitors lacking sufficient NaV1.7 selectivity over NaV1.6 were associated with respiratory cessation after in vivo administration to rodents. Effects on respiratory rate in rats were consistent with effects in an ex vivo hemisected rat diaphragm model and in vitro NaV1.6 potency. Furthermore, direct blockade of the phrenic nerve signaling was observed at exposures known to cause respiratory cessation in rats. Collectively, these results support a significant role for NaV1.6 in phrenic nerve signaling and respiratory function.

Ultrasound Analysis of Respiration-Related Muscles in Rats.

The purpose of this study was to evaluate the effectiveness of ultrasound techniques in the analysis of respiratory-related muscles in rats. Respiratory parameters, including diaphragm end-expiratory thickness, mean rectus abdominis (RA) thickness, and RA area, were measured by ultrasound and compared with histological findings. Spearman's correlation and Logistic regression analysis were used to detect the differences in the correlation between ultrasound results and histological examinations, and Student's t test was used to compare the differences between ultrasound results and histological examination data. The results showed that there was no significant difference between the end-expiratory thickness of the diaphragm, the average thickness of RA, and the area of RA in the right RA and histological values under ultrasound detection (p > 0.05), but there was a significant positive correlation between ultrasound, and histological values (p < 0.05).); in addition, tidal volume was significantly positively correlated with total RA area, rapid shallow breathing index (RSBI) was significantly negatively correlated with total RA area, and mean diaphragm TF was significantly positively correlated with tidal volume. In conclusion, ultrasound imaging has a high degree of accuracy and reproducibility and can be used to assess the structure and function of the rat diaphragm and RA.

Influence of moderate and high intensity interval training on infarcted rat diaphragm

Abstract Background Respiratory muscle dysfunction is commonly observed in heart failure (HF) patients and contributes to early fatigue and dyspnea. As there is no specific pharmacological therapy to treat skeletal muscle changes, physical exercise is considered an important strategy to prevent HF-associated skeletal myopathy. However, which exercise type is better during HF is unsettled. Purpose To evaluate the effects of aerobic and high intensity interval (HIIT) training on diaphragm morphometric, biochemical, and molecular parameters in infarcted rats. Methods Three months after inducing myocardial infarction (MI), male Wistar rats were assigned into three groups: sedentary MI (MI-Sed, n=8), MI subjected to aerobic exercise (MI-A, n=8), and MI subjected to HIIT (MI-HIIT, n=8). Results were compared to a Sham group (n=8). Exercise was performed in treadmill three times a week for three months. Functional capacity test and echocardiogram were performed before and after exercise protocols. Myosin heavy chain (MyHC) isoforms were analyzed by electrophoresis; diaphragm mitogen activated protein kinases (MAPK), NF-κB, TNF-α, and Pax-7 protein expression was evaluated by Western-blot. Statistical analysis: ANOVA. Results Functional capacity was similarly improved by both exercise protocols. Infarction size evaluated by histological analysis did not differ between groups (MI-Sed 27.7±10.8; MI-A 29.6±12.9; MI-HIIT 27.4±12.2% of total left ventricle area; p=0.91). Echocardiographic parameters did not differ between MI groups before and after exercise. Cross sectional area of diaphragm fibers (Sham 1922±230; MI-Sed 1808±264; MI-A 1926±209; MI-HIIT 2007±286 μm2), collagen interstitial fractional area (Sham 4.93±1.76; MI-Sed 5.01±1.88; MI-A 4.55±1.18; MI-HIIT 3.94±1.15%), and percentage of MyHC isoforms did not differ between groups. Pax-7 expression was lower in MI-HIIT than MI-Sed and MI-A [Sham 1.15 (0.58–1.44), MI-Sed 1.19 (0.90–2.85), MI-A 1.46 (0.77–2.14), MI-HIIT 0.46 (0.29–0.66) arbitrary units]. Total p-38 expression was higher in MI-Sed than the other groups and lower in MI-HIIT than Sham [Sham 1.00±0.52; MI-Sed 1.77±0.47; MI-A 0.66±0.22; MI-HIIT 0.46±0.31 arbitrary units]. The other proteins did not differ between groups. Conclusion Both aerobic and high intensity interval training improves functional capacity with no influence on diaphragm of rats with myocardial infarction. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): FAPESP (São Paulo Research Foundation)

Chronic Ouabain Prevents Radiation-Induced Reduction in the α2 Na,K-ATPase Function in the Rat Diaphragm Muscle

The damaging effect of ionizing radiation (IR) on skeletal muscle Na,K-ATPase is an open field of research. Considering a therapeutic potential of ouabain, a specific ligand of the Na,K-ATPase, we tested its ability to protect against the IR-induced disturbances of Na,K-ATPase function in rat diaphragm muscle that co-expresses the α1 and α2 isozymes of this protein. Male Wistar rats (n = 26) were subjected to 6-day injections of vehicle (0.9% NaCl) or ouabain (1 µg/kg/day). On the fourth day of injections, rats were exposed to one-time total-body X-ray irradiation (10 Gy), or a sham irradiation. The isolated muscles were studied 72 h post-irradiation. IR decreased the electrogenic contribution of the α2 Na,K-ATPase without affecting its protein content, thereby causing sarcolemma depolarization. IR increased serum concentrations of ouabain, IL-6, and corticosterone, decreased lipid peroxidation, and changed cellular redox status. Chronic ouabain administration prevented IR-induced depolarization and loss of the α2 Na,K-ATPase electrogenic contribution without changing its protein content. This was accompanied with an elevation of ouabain concentration in circulation and with the lack of IR-induced suppression of lipid peroxidation. Given the crucial role of Na,K-ATPase in skeletal muscle performance, these findings may have therapeutic implications as countermeasures for IR-induced muscle pathology.

Effect Of Fruit And Cork Extract Of Ficus Lacor Buch Ham On α/β -Glucosidase, α -Amylase, Lipase, Glucose Absorption And Uptake

Fruits of the plant Ficus Lacor Buch. Ham. were used traditionally for treatment of diabetes mellitus. The present study was undertaken to evaluate the antidiabetic potential of the plant using in vitro approach. Effect of Ficus Lacor Buch. Ham. was evaluated using α/β -glucosidase, α-amylase and lipase enzyme inhibition assay methods. The glucose absorption in intestine was evaluated using everted rat jejunum while glucose uptake was evaluated using isolated rat hemidiaphragm. Fruit and cork ethanolic extract was prepared by using soxhlation extraction method. In vitro assay of α-glucosidase showed that IC50 value of fruit extract was 83.03 µg/ml and cork extract 88.32 µg/ml when compared with control group acarbose. β-glucosidase enzyme was inhibited by fruit and cork extract of plant with IC50 value of fruit and cork extract 132.71 µg/ml and 171.93 µg/ml. The extracts further quantify α-amylase inhibitory activity of fruit (IC50 77.93 µg/ml) and cork (IC50 111.94 µg/ml) extract. Lipase inhibitory assay indicated the effect of plant extract on lipase enzyme was not prominent when compared to orlistat. Absorption of glucose through everted rat jejunum was reduced significantly (P ? 0.05) when compared with standard metformin. Effect of fruit and cork extract on rat hemidiaphragm exhibited significant (P ? 0.05) increase in glucose uptake when compared with standard metformin. Result suggests Ficus Lacor Buch. Ham. is effective in inhibiting carbohydrate metabolizing enzymes α/β –glucosidase and α-amylase while lipase enzyme was not affected. Fruit and cork extract of the plant was found to reduce significantly glucose absorption in everted rat jejunum. The significant increase in glucose uptake was observed in isolated rat diaphragm. The result reveals that Ficus Lacor Buch. Ham. acts by inhibiting carbohydrate metabolizing enzymes, reducing glucose absorption in intestine and increasing glucose uptake in hemidiaphragm.

Neuropathology of distinct diaphragm areas following mid-cervical spinal cord contusion in the rat

BACKGROUNDThe diaphragm is innervated by phrenic motoneurons distributed from the third to fifth cervical spinal cord. The rostral to caudal phrenic motoneuron pool segmentally innervates the ventral, medial, and dorsal diaphragm. PURPOSEThe present study was designed to investigate the physiological and transcriptomic mechanism of neuropathology of distinct diaphragm areas following mid-cervical spinal cord injury. STUDY DESIGNIn vivo animal study. METHODSElectromyograms and transcriptome of the ventral, medial, and dorsal diaphragm were examined in rats that received cervical laminectomy or mid-cervical spinal cord contusion in the acute (ie, 1–3 days) or subchronic (ie, ∼14 days) injury stages. RESULTSMid-cervical spinal cord contusion significantly attenuated the inspiratory bursting amplitude of the dorsal diaphragm but not the ventral or medial diaphragm. Moreover, the discharge onset of the dorsal diaphragm was significantly delayed compared with that of the ventral and medial diaphragm in contused rats. Transcriptomic analysis revealed a robust change in gene expression in the ventral diaphragm compared with that in the dorsal diaphragm. Specifically, enrichment analysis of differentially expressed genes demonstrated that the cell cycle and immune response were significantly upregulated, whereas several metabolic pathways were downregulated, in the ventral diaphragm of acutely contused rats. However, no significant Kyoto Encyclopedia of Genes and Genomes pathway was altered in the dorsal diaphragm. CONCLUSIONSThese results suggest that mid-cervical spinal cord injury has different impacts on the physiological and transcriptomic responses of distinct diaphragm areas. CLINICAL SIGNIFICANCEFuture therapeutic strategies can consider applying different therapies to distinct diaphragm areas following cervical spinal cord injury. Additionally, confirmation of activities across different diaphragm areas may provide a critical reference for the placement of diaphragmatic pacing electrodes.

Age‐dependent differences in the ventilator‐induced diaphragm dysfunction (VIDD): A multi‐omics study

Introduction &amp; ObjectiveMechanical ventilation is a life‐saving intervention in patients with respiratory failure. Controlled mechanical ventilation (CMV) is the mode of mechanical ventilation used in patients with an insufficient central respiratory drive and frequently used in COVID‐19 patients ventilated in the prone position. However, CMV is also associated with negative consequences for the major respiratory muscle the diaphragm, i.e., the ventilator induced diaphragm dysfunction (VIDD). VIDD is the major factor underlying the delayed weaning from the ventilator especially in old intensive care unit (ICU) patients. The age‐related difference in mortality rate has become a major concern during the COVID‐19 pandemic. The current experimental study was undertaken to improve our understanding the mechanisms underlying age‐related differences in the response to CMV using a unique experimental ICU model allowing long‐term studies in a rat exposed CMV.Materials and MethodsYoung (7‐8 months) and old (28‐32 months) female F344 BN hybrid rats were exposed to the ICU condition for 5 days (anaesthetized, mechanically ventilated, and pharmacologically paralyzed by post‐synaptic blockade of the neuromuscular transmission). Transcriptomics (RNA‐Seq) and proteomics (Olink) analyses of the diaphragm and proteomics analysis of plasma were conducted to investigate the molecular differences between young and old rats exposed to the ICU condition.ResultsAccording to multi‐omics analyses, significant differences were observed in the diaphragm between young and old rats in response to ICU condition. In the diaphragm of young rats, CMV resulted in decreases in the biological processes related to energy metabolism and production as indicated by the downregulation of pathway like “glycolysis/gluconeogenesis”, “fatty acid metabolism”, “TCA cycle”, and “oxidative phosphorylation”, etc. On the other hand, in the diaphragm of old rats, dramatic immune and inflammatory responses were observed after CMV, as reflected in the upregulation of “Cytokine‐cytokine receptor interaction”, “TNF signaling pathway”, “IL‐17 signaling pathway”, etc. Transcriptomic results were consistent with plasma proteomics analyses, i.e., CMV induced a higher level of inflammatory factors and cytokines/chemokines in old rats.ConclusionsAfter 5‐days exposure to CMV, our multi‐omics study demonstrated age‐related inflammatory response to long‐term mechanical ventilation.Significance/ImplicationThus, CMV should be cautiously used and restricted to as short duration as possible, especially in the old patients. In the current COVID‐19 pandemic, the dramatically increased mortality in old ICU patients with COVID‐19‐associated hyperinflammation and cytokine storm need not only reflect the viral infection but may also be associated with the VIDD and hyperinflammatory responses induced by CMV per se.

Cervical afferents are necessary for closed‐loop epidural stimulation‐induced respiratory neuroplasticity

Epidural electrical stimulation restores vital functions in neurological disorders such as stroke and spinal cord injury. After spinal injury, epidural stimulation improves locomotion, cardiovascular and bladder function, and trunk stability via neuromodulation of spinal neural networks. We have shown in rats that four days of epidural stimulation given via a closed‐loop paradigm (CLES) elicits spinal respiratory plasticity (Malone et al., FASEB J 2020;2021). Precise mechanisms of these effects in other sensorimotor systems are unclear, but modeling (Capogrosso et al., 2013) and rodent (Lavrov et a., 2008) studies suggest that inputs from segmental sensory afferent neurons are essential. We hypothesized that intact sensory afferents from the diaphragm (e.g. C3‐C5) are necessary for CLES‐induced respiratory plasticity. To test this, adult, female, Sprague‐Dawley rats were implanted with CLES electrodes at C4 and bilateral diaphragm recording electrodes (n=14). A subset of rats received C3‐C5 cervical dorsal rhizotomy (CDR) at the time of electrode implantation (n=6). After 1 week of recovery, rats received CLES (sub‐motor threshold epidural stimulation triggered by diaphragm EMG) ~20hr per day for 14 days. Inspiratory‐triggered spinal motor evoked potentials were recorded every 2 days. CLES for 2 weeks facilitated left diaphragm peak to peak amplitude of the stimulus‐triggered average in sham rats on all days measured (vs. day 0; all p&lt; 0.05). Right diaphragm in sham animals only showed facilitation on day 12 vs day 0 (0.005 vs 0.003; p&lt;0.05). In contrast, CDR rats exhibited blunted peak to peak magnitudes vs sham on all days except day 6 in the left diaphragm where there was also facilitation (vs day 0; p&lt;0.05). Days 2, 4, 12 and 14 of peak to peak magnitude in the right diaphragm of CDR rats was significantly lower than sham (all p&lt;0.05) and vs day 0 (all p&lt;0.05). In addition, motor threshold (e.g. the lowest current that can elicit an evoked potential) was significantly higher in CDR vs sham rats on days 4 (14.27% ± 15.02%; p=0.005), 6 (19.23% ± 21.89% vs ‐29.43% ± 3.77%; p=0.003), and 8 (18.10% ± 16.05% vs ‐20.25% ± 10.34%; p=0.004). These results suggest that CDR may attenuate the magnitude of the evoked potential and capacity to facilitate over the course of 14 days. However, it appears some compensatory plasticity may be occurring after CDR since 1) the increase in motor thresholds return to what is seen in sham animals after day 10 of CLES and 2) facilitation of the stimulus triggered average was seen on day 6 in the left diaphragm. It is also interesting to note the differential effects of CLES on right vs. left diaphragm, highlighting the differences in innervation or possible laterality in the capacity for spinal plasticity in this setting. Ongoing immunohistochemical analyses of C3‐C5 spinal cord and diaphragm may lend more insight into the mechanistic underpinnings of these results. This work represents the first report that long‐term (i.e. 2 weeks) CLES can elicit spinal respiratory neuroplasticity in healthy, awake, freely‐behaving rats and that intact sensory afferents may be necessary for this to occur. Future studies will be essential to elucidate the therapeutic potential of neuromodulation for improvement of respiratory deficits in individuals with neurological disorders.