Denervation versus pre- and postsynaptic muscle immobilization: Effects On acetylcholine- and muscle-specific tyrosine kinase receptors.

INTRODUCTION: Functional immobility of the diaphragm by mechanical ventilation impairs neuromuscular transmission and may result in ventilator-induced diaphragmatic dysfunction. We compared 3 diaphragmatic immobilization models with respect to their effects on expression of adult and fetal acetylcholine receptors (AChRs), muscle-specific receptor tyrosine kinase (MuSK), and muscle fiber morphology. METHODS: Diaphragms of rats were immobilized by either: (1) phrenicotomy; (2) presynaptic tetrodotoxin nerve blockade; or (3) postsynaptic polyethylene orthosis. AChR subtypes and MuSK were quantified by Western blot and immunohistochemistry. Muscle fiber morphology was evaluated by hematoxylin-eosin staining. RESULTS: Adult AChRs remained unchanged, whereas fetal AChRs and MuSK were upregulated in all models. Denervation induced the strongest changes in muscle morphology. CONCLUSIONS: Each diaphragm immobilization model led to severe morphologic and postsynaptic receptor changes. Postsynaptic polyethylene orthosis, a new model with an intact and functioning motor unit, best reflects the clinical picture of a functionally immobilized diaphragm. Muscle Nerve 55: 101-108, 2017.

Development of an algorithm using clinical tests to avoid post-operative residual neuromuscular block.

BACKGROUND: Quantitative neuromuscular monitoring is the gold standard to detect postoperative residual curarization (PORC). Many anesthesiologists, however, use insensitive, qualitative neuromuscular monitoring or unreliable, clinical tests. Goal of this multicentre, prospective, double-blinded, assessor controlled study was to develop an algorithm of muscle function tests to identify PORC. METHODS: After extubation a blinded anesthetist performed eight clinical tests in 165 patients. Test results were correlated to calibrated electromyography train-of-four (TOF) ratio and to a postoperatively applied uncalibrated acceleromyography. A classification and regression tree (CART) was calculated developing the algorithm to identify PORC. This was validated against uncalibrated acceleromyography and tactile judgement of TOF fading in separate 100 patients. RESULTS: After eliminating three tests with poor correlation, a model with four tests (r = 0.844) and uncalibrated acceleromyography (r = 0.873) were correlated to electromyographical TOF-values without losing quality of prediction. CART analysis showed that three consecutively performed tests (arm lift, head lift and swallowing or eye opening) can predict electromyographical TOF. Prediction coefficients reveal an advantage of the uncalibrated acceleromyography in terms of specificity to identify the EMG measured train-of-four ratio < 0.7 (100% vs. 42.9%) and <0.9 (89.7% vs. 34.5%) compared to the algorithm. However, due to the high sensitivity of the algorithm (100% vs. 94.4%), the risk to overlook an awake patient with a train-of-four ratio < 0.7 was minimal. Tactile judgement of TOF fading showed poorest sensitivity and specifity at train of four ratio < 0.9 (33.7%, 0%) and <0.7 (18.8%, 16.7%). CONCLUSIONS: Residual neuromuscular blockade can be detected by uncalibrated acceleromyography and if not available by a pathway of four clinical muscle function tests in awake patients. The algorithm has a discriminative power comparable to uncalibrated AMG within TOF-values >0.7 and <0.3. TRIAL REGISTRATION: Clinical Trials.gov (principal investigator's name: CU, and identifier: NCT03219138) on July 8, 2017.

Neuromuscular Recovery Is Prolonged After Immobilization or Superimposition of Inflammation With Immobilization Compared to Inflammation Alone: Data From a Preclinical Model.

OBJECTIVES: Recovery from ICU-acquired muscle weakness extends beyond hospital stay. We hypothesized that immobilization, more than inflammation, plays a prominent role in the delayed recovery from critical illness. DESIGN: Prospective, randomized, controlled, experimental study. SETTING: Animal laboratory, university hospital. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: Animals were divided to have one hind limb immobilized (n = 129) or sham-immobilized (n = 129) on day -12. After surgery, rats were further assigned to two subgroups. To induce inflammation, rats received three IV injections of Corynebacterium parvum on days -12, -8, and -4. Controls received saline at the respective time-points. At day 0, the limbs were remobilized and recovery from inflammation and/or immobilization was followed for 36 days. MEASUREMENTS AND MAIN RESULTS: At day 0 and after 4, 12, or 36 days of recovery, maximum tetanic tension and tetanic fade (functional parameters = primary outcome variables) as well as nicotinic acetylcholine receptor expression, muscle mass, and histologic changes (structural parameters = secondary outcome variables) were measured. Impaired maximum tetanic tension, decreased tibialis muscle mass, and fiber diameter due to inflammation alone recovered by day 4. Tetanic fade was not affected by inflammation. Immobilization-induced loss of tibialis muscle mass, decreased fiber diameter, and tetanic fade did not return to normal until day 36, while maximum tetanic tension had recovered at that time. In the presence of inflammation and immobilization, the decrease in tibialis muscle mass, fiber diameter, and maximum tetanic tension, as well as decreased tetanic fade persisted until day 36. Up-regulation of nicotinic acetylcholine receptors normalized before day 4 following inflammation, but persisted until day 4 following immobilization. CONCLUSIONS: In our model, muscle function and structure recovered from inflammation within 4-12 days. Immobilization-induced neuromuscular changes, however, persisted even at day 36, especially if inflammation was concomitant.

Neuromuscular blockade improves surgical conditions (NISCO).

BACKGROUND: We examined the impact of muscle relaxation on surgical conditions and patients' postoperative outcome during elective laparoscopic cholecystectomy under balanced anaesthesia. METHODS: After approval and consent, 57 anaesthetized patients were randomly assigned to group no neuromuscular blockade (No NMB) and deep neuromuscular blockade (Deep NMB), i.e. no twitch response to train-of-four nerve stimulation. Laparoscopic cholecystectomy was performed using the 4-trocar technique with a CO2-pneumoperitoneum. Surgical conditions were assessed using a Visual Analogue Scale. Movement of diaphragm or abdominal muscles, inadequate visibility, or breathing and coughing against the ventilator were documented as events reflecting inadequate muscle relaxation. Independently, surgeons could request 0.3 mg/kg rocuronium to improve surgical conditions. Workflow variables were obtained as a surrogate of surgical conditions. Data are presented as mean (95 % confidence interval). The trial is registered at ClinicalTrials.gov (NCT00895778). RESULTS: While in 12 of 25 patients of group "No NMB" one or more adverse events impaired the surgical procedure (p < 0.001), only 1 of 25 patients of group "Deep NMB" showed an adverse event. Deep NMB resulted in an absolute risk reduction of 0.44 (0.23-0.65) and a number needed to treat of 2.3 (1.5-4.4), respectively. Surgeons requested 0.3 mg/kg rocuronium in 10 of 25 cases (40 %) of group "No NMB" only. This dose significantly improved surgical conditions by an average 62 of 100 possible points. All further variables did not differ between groups. CONCLUSIONS: Deep NMB ameliorates surgical conditions for laparoscopic cholecystectomy by improved visibility and reduction of involuntary movements.

Prolonged administration of pyridostigmine impairs neuromuscular function with and without down-regulation of acetylcholine receptors.

BACKGROUND: The acetylcholinesterase inhibitor, pyridostigmine, is prophylactically administered to mitigate the toxic effects of nerve gas poisoning. The authors tested the hypothesis that prolonged pyridostigmine administration can lead to neuromuscular dysfunction and even down-regulation of acetylcholine receptors. METHODS: Pyridostigmine (5 or 25 mg·kg·day) or saline was continuously administered via osmotic pumps to rats, and infused for either 14 or 28 days until the day of neuromuscular assessment (at day 14 or 28), or discontinued 24 h before neuromuscular assessment. Neurotransmission and muscle function were examined by single-twitch, train-of-four stimulation and 100-Hz tetanic stimulation. Sensitivity to atracurium and acetylcholine receptor number (quantitated by I-α-bungarotoxin) provided additional measures of neuromuscular integrity. RESULTS: Specific tetanic tensions (Newton [N]/muscle weight [g]) were significantly (P < 0.05) decreased at 14 (10.3 N/g) and 28 (11.1 N/g) days of 25 mg·kg·day pyridostigmine compared with controls (13.1-13.6 N/g). Decreased effective dose (0.81-1.05 vs. 0.16-0.45 mg/kg; P < 0.05) and decreased plasma concentration (3.02-3.27 vs. 0.45-1.37 µg/ml; P < 0.05) of atracurium for 50% paralysis (controls vs. 25 mg·kg·day pyridostigmine, respectively), irrespective of discontinuation of pyridostigmine, confirmed the pyridostigmine-induced altered neurotransmission. Pyridostigmine (25 mg·kg·day) down-regulated acetylcholine receptors at 28 days. CONCLUSIONS: Prolonged administration of pyridostigmine (25 mg·kg·day) leads to neuromuscular impairment, which can persist even when pyridostigmine is discontinued 24 h before assessment of neuromuscular function. Pyridostigmine has the potential to down-regulate acetylcholine receptors, but induces neuromuscular dysfunction even in the absence of receptor changes.

Intracellular serotonin modulates insulin secretion from pancreatic beta-cells by protein serotonylation.

While serotonin (5-HT) co-localization with insulin in granules of pancreatic beta-cells was demonstrated more than three decades ago, its physiological role in the etiology of diabetes is still unclear. We combined biochemical and electrophysiological analyses of mice selectively deficient in peripheral tryptophan hydroxylase (Tph1-/-) and 5-HT to show that intracellular 5-HT regulates insulin secretion. We found that these mice are diabetic and have an impaired insulin secretion due to the lack of 5-HT in the pancreas. The pharmacological restoration of peripheral 5-HT levels rescued the impaired insulin secretion in vivo. These findings were further evidenced by patch clamp experiments with isolated Tph1-/- beta-cells, which clearly showed that the secretory defect is downstream of Ca(2+)-signaling and can be rescued by direct intracellular application of 5-HT via the clamp pipette. In elucidating the underlying mechanism further, we demonstrate the covalent coupling of 5-HT by transglutaminases during insulin exocytosis to two key players in insulin secretion, the small GTPases Rab3a and Rab27a. This renders them constitutively active in a receptor-independent signaling mechanism we have recently termed serotonylation. Concordantly, an inhibition of such activating serotonylation in beta-cells abates insulin secretion. We also observed inactivation of serotonylated Rab3a by enhanced proteasomal degradation, which is in line with the inactivation of other serotonylated GTPases. Our results demonstrate that 5-HT regulates insulin secretion by serotonylation of GTPases within pancreatic beta-cells and suggest that intracellular 5-HT functions in various microenvironments via this mechanism in concert with the known receptor-mediated signaling.

A single injection of botulinum toxin decreases the margin of safety of neurotransmission at local and distant sites.

BACKGROUND: We tested the hypothesis that a single injection of botulinum toxin not only has local, but also distant effects on muscle function, biochemistry, and pharmacodynamics of atracurium. METHODS: Botulinum toxin (2.5 U) was injected into the tibialis muscle of anesthetized rats (n = 26). The contralateral side with no injection served to study distant effects. Control animals (n = 25) received a saline injection. Neuromuscular function, pharmacology, and expression of acetylcholine receptors (nAChRs) were evaluated in the tibialis at 0, 4, and 16 days after injection and in comparison with saline- injected controls. RESULTS: On day 4, botulinum toxin caused complete paralysis of the tibialis, while its contralateral side showed a decrease in absolute twitch tension (1.8 N [1.6; 1.9] vs 3.0 N [2.8; 3.1], Newton, P < 0.05). On day 16, muscle weakness was only present on the toxin-injected side where absolute twitch tension was decreased (0.6 N [0.6, 0.7] vs 3.4 N [3.1, 3.7], P < 0.05). Tibialis mass was decreased on the toxin-injected side at day 4 (1.46 mg/g [1.43, 1.48] vs 1.74 mg/g [1.72; 1.75], P < 0.05) and on day 16 (0.78 mg/g [0.76, 0.79] vs 1.73 mg/g [1.69; 1.77], P < 0.05). Effects distant from the site of injection were seen on day 16, when muscle atrophy was also present in the adjacent gastrocnemius and soleus muscles. Normalized to tibialis mass, specific twitch tension (tension/g muscle) was reduced on the contralateral side at day 4 and on the toxin-injected side at day 16 in relation to saline controls. At day 16, an increased sensitivity to atracurium was seen on the toxin-injected side, evidenced as a decreased ED(50) (0.23 mg/kg [0.13, 0.33] vs 0.72 mg/kg [0.63, 0.82], P < 0.05) and a lower infusion rate (38 µL/kg/min [32, 43] vs135 µL/kg/min [126, 144], P < 0.05), together with a reduced plasma concentration requirement of atracurium (0.5 µg/mL [0.4, 0.7] vs 4.5 µg/mL [3.8, 5.2], P < 0.05) to achieve a steady state 50% reduction in baseline (absolute) twitch tension. ED(50) of atracurium was also decreased on the contralateral side at day 16 in relation to saline controls. The nAChRs in the tibialis were increased on the toxin-injected side to 123 fmol/mg [115, 131] vs 28 fmol/mg [25, 29] (P < 0.05) in time-matched saline-injected controls at day 4 and to 378 [341, 413] vs 27 fmol/mg [25, 29] (P < 0.05) at day 16. CONCLUSIONS: Botulinum toxin has local and distant effects on muscle. The decrease in specific twitch tension indicates that the muscle atrophy alone cannot explain the functional changes; neuromuscular transmission is also impaired. An increased sensitivity to atracurium on the toxin-injected side, despite up-regulation of nAChRs, seems unique to botulinum toxin.

The integrity of cholinergic basal forebrain neurons depends on expression of Nkx2-1.

The transcription factor Nkx2-1 belongs to the homeobox-encoding family of proteins that have essential functions in prenatal brain development. Nkx2-1 is required for the specification of cortical interneurons and several neuronal subtypes of the ventral forebrain. Moreover, this transcription factor is involved in migratory processes by regulating the expression of guidance molecules. Interestingly, Nkx2-1 expression was recently detected in the mouse brain at postnatal stages. Using two transgenic mouse lines that allow prenatal or postnatal cell type-specific deletion of Nkx2-1, we show that continuous expression of the transcription factor is essential for the maturation and maintenance of cholinergic basal forebrain neurons in mice. Notably, prenatal deletion of Nkx2-1 in GAD67-expressing neurons leads to a nearly complete loss of cholinergic neurons and parvalbumin-containing GABAergic neurons in the basal forebrain. We also show that postnatal mutation of Nkx2-1 in choline acetyltransferase-expressing cells causes a striking reduction in their number. These degenerative changes are accompanied by partial denervation of their target structures and results in a discrete impairment of spatial memory.

Continuous administration of pyridostigmine improves immobilization-induced neuromuscular weakness.

OBJECTIVE: To investigate the effects of continuous pyridostigmine infusion on immobilization-induced muscle weakness. Critical illness often results in immobilization of limb and respiratory muscles, leading to muscle atrophy and up-regulation of nicotinic acetylcholine receptors. Pyridostigmine reversibly blocks acetylcholinesterase and has the potential to improve neuromuscular transmission and decrease acetylcholine receptor number. DESIGN: Prospective, randomized, controlled experimental study. SETTING: Animal laboratory, university hospital. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: A total of 40 rats were immobilized in one hind limb by pinning knee and ankle joints. Rats received either continuous pyridostigmine (15 mg.kg.day) or saline subcutaneously via implanted osmotic pumps. MEASUREMENTS AND MAIN RESULTS: After 7 days and 14 days of immobilization, neuromuscular function, atracurium pharmacodynamics, and expression of acetylcholine receptors were evaluated. At 7 days and 14 days after immobilization, muscle force decreased in all untreated groups, whereas effective doses for paralysis with atracurium and acetylcholine receptor number in the tibialis were significantly increased. Pyridostigmine-treated rats showed a significantly improved muscle force and muscle mass in the immobilized limb. This was associated with an attenuation of acetylcholine receptor up-regulation in the respective leg. At this time, the dose-response curve for atracurium on the immobilized side was shifted to the left in the pyridostigmine group. After 14 days, muscle tension was still less depressed with pyridostigmine infusion, and resistance to the effects of atracurium was still attenuated. However, there were no differences in acetylcholine receptor expression between the immobilized sides of both groups. CONCLUSIONS: Continuous pyridostigmine infusion improves muscle weakness after 7 days and 14 days of immobilization. The up-regulation of acetylcholine receptors and the concomitant resistance to atracurium is attenuated in animals treated with pyridostigmine after 7 days of immobilization.

Sugammadex and neostigmine dose-finding study for reversal of shallow residual neuromuscular block.

INTRODUCTION: Sugammadex effectively and rapidly reverses deep to moderate rocuronium-induced neuromuscular block. However, the required dose of sugammadex for smaller degrees of residual block is unknown. Therefore we investigated the efficacy of sugammadex and neostigmine at a train-of-four (TOF) ratio of 0.5. METHODS: After ethics committee (Munich, Germany) approval and written informed consent were obtained, 99 patients were anesthetized with propofol, remifentanil, and rocuronium. Neuromuscular monitoring was performed by calibrated electromyography. At recovery of the TOF ratio to 0.5, patients randomly received sugammadex (0.0625, 0.125, 0.25, 0.5, or 1.0 mg/kg), neostigmine (5, 8, 15, 25, or 40 µg/kg), or saline. The time between study drug injection, at TOF ratio of 0.5, and postoperative TOF ratio of 0.9 was measured. The dose-response relationship was analyzed with a biexponential model using the dose as the independent variable and the logarithm of the recovery time as the dependent variable. Effective doses were interpolated from regression models. RESULTS: Sugammadex, 0.22 mg/kg, is able to reverse a TOF ratio of 0.5 to 0.9 or higher in an average time of 2 min. Within 5 min, 95% of patients reach this TOF ratio. Neostigmine, 34 µg/kg, is able to reverse a TOF ratio of 0.5 to 0.9 or higher within 5 min. No recurarization was observed. CONCLUSIONS: Sugammadex, 0.22 mg/kg, and neostigmine, 34 µg/kg, effectively and comparably reverse a rocuronium-induced shallow residual neuromuscular block at a TOF ratio of 0.5.

Altered nicotinamide adenine dinucleotide (NADH) fluorescence in dt sz mutant hamsters reflects differences in striatal metabolism between severe and mild dystonia.

The dt(sz) mutant hamster represents a unique rodent model of idiopathic paroxysmal dystonia. Previous data, collected post-mortem or in anesthetized hamsters under basal conditions, indicated the critical involvement of enhanced striatal neuronal activity. To assess the importance of an enhanced striatal neuronal activity directly during a dystonic episode, continuous monitoring of changes in brain metabolism and therefore neuronal activity indirectly in awake, freely moving animals is necessary. Determination of CNS metabolism by NADH measurement by laser-induced fluorescence spectroscopy in conscious dt(sz) and nondystonic control hamsters revealed reversible decreased NADH fluorescence during dystonic episodes. The degree of change corresponded to the severity of dystonia. This study represents the first application of this innovative method in freely moving animals exhibiting a movement disorder. Our data clearly confirm that the expression of paroxysmal dystonia in dt(sz) mutant hamsters is associated with enhanced striatal neuronal activity and further underscore the versatile application of NADH fluorescence measurements in neuroscience.

Increasing the number of 5-HT(1A)-receptors in cortex and hippocampus does not induce mnemonic deficits in mice.

Even though the role of the serotonin1A (5-HT(1A))-receptor for cognitive processes is undisputed, the exact involvement of pre- and postsynaptic sites remains unexplained. Recently, we introduced a mouse line overexpressing the 5-HT(1A)-receptor in the hippocampus and cortex. In this study we investigated in comparison to wild-type mice their cognitive abilities using the Morris water-maze task and inhibitory avoidance test. Acute effects of pre- and posttraining administered 8-OH-DPAT (0.03-0.3 mg/kg i.p.) were examined in the inhibitory avoidance test. Additionally, habituation learning was studied in the hole-board test. Transgenic mice showed no overall learning deficit. Spatial learning and memory revealed in the Morris water-maze task was comparable to wild-type mice, and both genotypes habituated to the hole-board arena in a similar manner. Comparing the performance of both genotypes in the inhibitory avoidance test, cognitive functions of transgenic mice seemed to be slightly impaired. When 8-OH-DPAT was administered pretraining an amnesic effect was produced only in transgenic mice and only at the highest dose (0.3 mg/kg). Posttraining administered 0.3 mg/kg 8-OH-DPAT did not affect the performance of both genotypes. Overall, the cortical and hippocampal overexpression of the 5-HT(1A)-receptor had no major effect on cognitive functions in mice, suggesting that changes in the 5-HT(1A)-receptor density are not necessarily accompanied with alterations of learning and memory processes.

Therapeutic efficacy of the Ginkgo special extract EGb761® within the framework of the mitochondrial cascade hypothesis of Alzheimer's disease.

OBJECTIVES: The mitochondrial cascade hypothesis of dementia assumes mitochondrial dysfunction as an important common pathomechanism for the whole spectrum of age-associated memory disorders from cognitive symptoms in the elderly over mild cognitive impairment to Alzheimer's dementia. Thus, a drug such as the Ginkgo special extract EGb 761® which improves mitochondrial function should be able to ameliorate cognitive deficits over the whole aging spectrum. METHODS: We review the most relevant publications about effects of EGb 761® on cognition and synaptic deficits in preclinical studies as well as on cognitive deficits in man from aging to dementia. RESULTS: EGb 761® improves mitochondrial dysfunction and cognitive impairment over the whole spectrum of age-associated cognitive disorders in relevant animal models and in vitro experiments, and also shows clinical efficacy in improving cognition over the whole range from aging to Alzheimer's or even vascular dementia. CONCLUSIONS: EGb 761® shows clinical efficacy in the treatment of cognitive deficits over the whole spectrum of age-associated memory disorders. Thus, EGb 761® can serve as an important pharmacological argument for the mitochondrial cascade hypothesis of dementia.

A mouse model for intellectual disability caused by mutations in the X-linked 2'­O­methyltransferase Ftsj1 gene.

Mutations in the X chromosomal tRNA 2'­O­methyltransferase FTSJ1 cause intellectual disability (ID). Although the gene is ubiquitously expressed affected individuals present no consistent clinical features beyond ID. In order to study the pathological mechanism involved in the aetiology of FTSJ1 deficiency-related cognitive impairment, we generated and characterized an Ftsj1 deficient mouse line based on the gene trapped stem cell line RRD143. Apart from an impaired learning capacity these mice presented with several statistically significantly altered features related to behaviour, pain sensing, bone and energy metabolism, the immune and the hormone system as well as gene expression. These findings show that Ftsj1 deficiency in mammals is not phenotypically restricted to the brain but affects various organ systems. Re-examination of ID patients with FTSJ1 mutations from two previously reported families showed that several features observed in the mouse model were recapitulated in some of the patients. Though the clinical spectrum related to Ftsj1 deficiency in mouse and man is variable, we suggest that an increased pain threshold may be more common in patients with FTSJ1 deficiency. Our findings demonstrate novel roles for Ftsj1 in maintaining proper cellular and tissue functions in a mammalian organism.

Pharmacokinetic aspects of reduced nicotinamide adenine dinucleotide (NADH) in rats.

Nicotinamide adenine dinucleotide (NADH) plays a major role in cellular metabolism and mitochondrial dysfunction and is thought that NAD+/NADH decrease neuronal degeneration and improve behavioral deficits. This potential use of NAD+ or NADH as neuroprotective drugs requires an insight on the pharmacokinetic properties of these compounds. For this reason, we assessed the absorption of NADH in the small intestine in vitro using the everted gut sac technique. We show an enteral absorption of the intact NADH molecule. In the gut sac, NADH had a concentration-independent absorption rate of about 5 percent and the in vivo laser-induced fluorescence spectroscopy revealed a relatively quick absorption of NADH starting after a few minute reaching a plateau (about 5 percent ) after 20-30 minutes. Theses results show that, should NADH be protected against the acidic conditions of the stomach, NADH is absorbed principally in the small intestine.

Systemic inflammatory response syndrome increases immobility-induced neuromuscular weakness.

OBJECTIVE: Inflammation and immobility are comorbid etiological factors inducing muscle weakness in critically ill patients. This study establishes a rat model to examine the effect of inflammation and immobilization alone and in combination on muscle contraction, histology, and acetylcholine receptor regulation. DESIGN: Prospective, randomized, experimental study. SETTING: Animal laboratory of a university hospital. SUBJECTS: Sprague-Dawley rats. INTERVENTIONS: To produce systemic inflammation, rats (n = 34) received three consecutive intravenous injections of Corynebacterium parvum on days 0, 4, and 8. Control rats (n = 21) received saline. Both groups were further divided to have one hind limb either immobilized by pinning of knee and ankle joints or sham-immobilized (surgical leg). The contralateral nonsurgical leg of each animal served as control (nonsurgical leg). MEASUREMENTS AND MAIN RESULTS: After 12 days, body weight and muscle mass were significantly reduced in all C. parvum animals compared with saline-injected rats. Immobilization led to local muscle atrophy. Normalized to muscle mass, tetanic contraction was reduced in the surgical leg after immobilization (7.64 +/- 1.91 N/g) and after inflammation (8.71 +/- 2.0 N/g; both p < .05 vs. sham immobilization and saline injection, 11.03 +/- 2.26 N/g). Histology showed an increase in inflammatory cells in all C. parvum-injected animals. Immobilization in combination with C. parvum injection had an additive effect on inflammation. Acetylcholine receptors were increased in immobilized muscles and in all muscles of C. parvum-injected animals. CONCLUSIONS: The muscle weakness in critically ill patients can be replicated in our novel rat model. Inflammation and immobilization independently lead to muscle weakness.

Chronic Escherichia coli infection induces muscle wasting without changing acetylcholine receptor numbers.

OBJECTIVE: Muscle weakness in septic patients is a serious problem as it complicates and lengthens hospital stays, prolongs rehabilitation and increases costs. We examined the effects of a chronic infection with Escherichia coli on muscle function, muscle mass, and the expression of nicotinic acetylcholine receptors (AChRs). DESIGN AND SETTING: Prospective, randomized animal study in an animal laboratory, university hospital. SUBJECTS: Sprague-Dawley rats. INTERVENTIONS: A catheter was implanted into the external jugular vein of anesthetized rats, and a dose of 3.2x10(8) CFU of E. coli bacteria was injected; the catheter was then sealed and tunneled subcutaneously. MEASUREMENTS AND RESULTS: Animals injected with E. coli bacteria showed a significant decrease in body and muscle mass over the 14-day experimental period. Neuromuscular function was tested by mechanomyography on days 3, 7, and 14 following injection. Tetanic tension decreased over the time course of sepsis, without effecting tetanic fade. Serum levels of acute-phase protein, alpha1-acid glycoprotein, were increased by day 3, and remained significantly higher until day 14. AChRs were quantitated using 125I-labeled bungarotoxin and revealed no differences between groups. CONCLUSIONS: Central venous injection of E. coli bacteria induces systemic inflammation evidenced as loss of body weight, muscle mass and increased alpha1-acid glycoprotein levels. The inflammation-induced muscle weakness is due to muscle atrophy and not to upregulated AChRs. This model may prove useful for studying maneuvers to prevent muscle wasting with inflammation.

Impact of repeated anesthesia with ketamine and xylazine on the well-being of C57BL/6JRj mice.

Within the scope of the 3Rs of Russel and Burch, the number of laboratory animals can be reduced by repeated use of an animal. This strategy only becomes relevant, if the total amount of pain, distress or harm the individual animal experiences does not exceed the severity of a single manipulation. For example, when using imaging techniques, an animal can be examined several times during a study, but it has to be anesthetized each time imaging is performed. The severity of anesthesia is thought to be mild according to the Directive 2010/63/EU. However, the Directive does not differentiate between single and repeated anesthesia, although repeated anesthesia may have a greater impact on well-being. Hence, we compared the impact of single and repeated anesthesia (six times at an interval of three to four days) by injection of ketamine and xylazine (KX) on the well-being of adult female and male C57BL/6JRj mice. After anesthesia, well-being of mice was assessed according to a protocol for systematic assessment of well-being including nesting, the Mouse Grimace Scale (MGS), a test for trait anxiety, home cage activity, and the rotarod test for motor activity, food intake, and body weight, as well as corticosterone (metabolite) analysis. Repeated anesthesia increased the MGS in mice of both sexes and caused short-term effects on well-being of female mice in the immediate post-anesthetic period, indicated by longer lasting effects on trait anxiety-related behavior. However, corticosterone metabolite concentrations suggested that mice habituated to the stress induced by repeated KX administration. Hence, the mildly negative effects on well-being of repeated KX anesthesia do not seem to accumulate over time using the respective regimen. However, further observations for severity classification are warranted in order to more specifically determine the duration of mild distress and trait anxiety.