Cold Water Immersion Offers No Functional or Perceptual Benefit Compared to a Sham Intervention During a Resistance Training Program.

ABSTRACT: Wilson, LJ, Dimitriou, L, Hills, FA, Gondek, MB, van Wyk, A, Turek, V, Rivkin, T, Villiere, A, Jarvis, P, Miller, S, Turner, A, and Cockburn, E. Cold water immersion offers no functional or perceptual benefit compared to a sham intervention during a resistance training program. J Strength Cond Res 35(10): 2720-2727, 2021-Cold water immersion (CWI) is regularly used by athletes as a postexercise recovery strategy, but relatively little is understood about potential training adaptations associated with habitual use. The aim of this study was to investigate the influence of repeated CWI or a sham intervention on adaptations to a lower body resistance training program. Thirteen men (26 ± 6 years; 83.6 ± 15.7 kg) familiar with resistance training were allocated into a CWI (10 minutes at 10° C) or sham group and completed 2 × 4-week blocks of lower body resistance training. Subjects completed a total of 16 training sessions (2 × session·week-1), with each session immediately followed by their allocated recovery intervention. Measures of perceptual markers, muscle function, and muscle architecture were recorded at baseline, midpoint, and post-training. Data were analyzed using factorial analysis of variances. The training program resulted in significant increases in muscle fibre pennation angle (p = 0.009), isometric peak force (p = 0.018), and 1/4 squat (p < 0.001) with no differences between groups (all p > 0.05). There were no differences in perceptual responses between groups. Despite the popularity of CWI as a postexercise recovery intervention, the findings from the present study demonstrated no functional or perceptual benefit compared with a sham intervention during progressive strength and power training. Furthermore, there was no detrimental impact of CWI on morphological adaptations after 16 exposures. These findings are important for athletes and practitioners wishing to use CWI as an acute recovery strategy after training, without blunting potential training adaptations.

Whole body cryotherapy, cold water immersion, or a placebo following resistance exercise: a case of mind over matter?

PURPOSE: The use of cryotherapy as a recovery intervention is prevalent amongst athletes. Performance of high volume, heavy load resistance exercise is known to result in disturbances of muscle function, perceptual responses and blood borne parameters. Therefore, this study investigated the influence of cold water immersion (CWI), whole body cryotherapy (WBC) or a placebo (PL) intervention on markers of recovery following an acute resistance training session. METHODS: 24 resistance trained males were matched into a CWI (10 min at 10 °C), WBC (3- and 4 min at - 85 °C) or PL group before completing a lower body resistance training session. Perceptions of soreness and training stress, markers of muscle function, inflammation and efflux of intracellular proteins were assessed before, and up to 72 h post exercise. RESULTS: The training session resulted in increased soreness, disturbances of muscle function, and increased inflammation and efflux of intracellular proteins. Although WBC attenuated soreness at 24 h, and positively influenced peak force at 48 h compared to CWI and PL, many of the remaining outcomes were trivial, unclear or favoured the PL condition. With the exception of CRP at 24 h, neither cryotherapy intervention attenuated the inflammatory response compared to PL. CONCLUSION: There was some evidence to suggest that WBC is more effective than CWI at attenuating select perceptual and functional responses following resistance training. However, neither cryotherapy intervention was more effective than the placebo treatment at accelerating recovery. The implications of these findings should be carefully considered by individuals employing cryotherapy as a recovery strategy following heavy load resistance training.

Analysis of procainamide-derivatised heparan sulphate disaccharides in biological samples using hydrophilic interaction liquid chromatography mass spectrometry.

Glycosaminoglycans (GAGs) are a family of linear heteropolysaccharides made up of repeating disaccharide units that are found on the surface and extracellular matrix of animal cells. They are known to play a critical role in a wide range of cellular processes including proliferation, differentiation and invasion. To elucidate the mechanism of action of these molecules, it is essential to quantify their disaccharide composition. Analytical methods that have been reported involve either chemical or enzymatic depolymerisation of GAGs followed by separation of non-derivatised (native) or derivatised disaccharide subunits and detection by either UV/fluorescence or MS. However, the measurement of these disaccharides is challenging due to their hydrophilic and labile nature. Here we report a pre-column LC-MS method for the quantification of GAG disaccharide subunits. Heparan sulphate (HS) was extracted from cell lines using a combination of molecular weight cutoff and anion exchange spin filters and digested using a mixture of heparinases I, II and III. The resulting subunits were derivatised with procainamide, separated using hydrophilic interaction liquid chromatography and detected using electrospray ionisation operated in positive ion mode. Eight HS disaccharides were separated and detected together with an internal standard. The limit of detection was found to be in the range 0.6-4.9 ng/mL. Analysis of HS extracted from all cell lines tested in this study revealed a significant variation in their composition with the most abundant disaccharide being the non-sulphated ∆UA-GlcNAc. Some structural functional relationships are discussed demonstrating the viability of the pre-column method for studying GAG biology. Graphical abstract Extraction and HILIC UPLC-MS analysis of procainamide-labelled heparan sulphate disaccharides.

Recovery following a marathon: a comparison of cold water immersion, whole body cryotherapy and a placebo control.

PURPOSE: Cryotherapy is an increasingly popular recovery strategy used in an attempt to attenuate the negative impact of strenuous physical activity on subsequent exercise. Therefore, this study aimed to assess the effects of whole body cryotherapy (WBC) and cold water immersion (CWI) on markers of recovery following a marathon. METHODS: Thirty-one endurance trained males completed a marathon. Participants were randomly assigned to a CWI, WBC or placebo group. Perceptions of muscle soreness, training stress and markers of muscle function were recorded before the marathon and at 24 and 48 h post exercise. Blood samples were taken at baseline, post intervention and 24 and 48 h post intervention to assess inflammation and muscle damage. RESULTS: WBC had a harmful effect on muscle function compared to CWI post marathon. WBC positively influenced perceptions of training stress compared to CWI. With the exception of C-reactive protein (CRP) at 24 and 48 h, neither cryotherapy intervention positively influenced blood borne markers of inflammation or structural damage compared to placebo. CONCLUSION: The findings show WBC has a negative impact on muscle function, perceptions of soreness and a number of blood parameters compared to CWI, contradicting the suggestion that WBC may be a superior recovery strategy. Further, cryotherapy is no more effective than a placebo intervention at improving functional recovery or perceptions of training stress following a marathon. These findings lend further evidence to suggest that treatment belief and the placebo effect may be largely responsible for the beneficial effects of cryotherapy on recovery following a marathon.

Heparan sulfate disaccharide measurement from biological samples using pre-column derivatization, UPLC-MS and single ion monitoring.

Glycosaminoglycans are a heterogeneous family of linear polysaccharides comprised of repeating disaccharide subunits that mediate many effects at the cellular level. There is increasing evidence that the nature of these effects is determined by differences in disaccharide composition. However, the determination of GAG disaccharide composition in biological samples remains challenging and time-consuming. We have developed a method that uses derivatization and selected ion recording and RP-UPLCMS resulting in rapid separation and quantification of twelve heparin/heparin sulfate disaccharides from 5 µg GAG. Limits of detection and quantitation were 0.02-0.15 and 0.07-0.31 µg/ml respectively. We have applied this method to the novel analysis of disaccharide levels extracted from heparan sulfate and human cancer cell lines. Heparan sulfate disaccharides extracted from biological samples following actinase and heparinase incubation and derivatized using reductive amination with 2-aminoacridone. Derivatized disaccharides were analyzed used UPLC-MS with single ion monitoring. Eight HS disaccharide subunits were separated and quantified from HS and cell lines in eleven minutes per sample. In all samples the most abundant subunits present were the unsulfated ΔUA-GlcNAc, ΔUA-GlcNAc,6S and ΔUA,2S-GlcNS,6S. There was considerable variation in the proportions and concentrations of disaccharides between different cell lines. Further studies are needed to examine the significance of these differences.

Competition Intensity and Fatigue in Elite Fencing.

Turner, AN, Kilduff, LP, Marshall, GJG, Phillips, J, Noto, A, Buttigieg, C, Gondek, M, Hills, FA, and Dimitriou, L. Competition intensity and fatigue in elite fencing. J Strength Cond Res 31(11): 3128-3136, 2017-As yet, no studies have characterized fencing competitions. Therefore, in elite male foilists and across 2 competitions, we investigated their countermovement jump height, testosterone (T), cortisol (C), alpha-amylase (AA), immunoglobulin A (IgA), heart rate (HR), blood lactate (BL), and rating of perceived exertion (RPE). Average (±SD) scores for RPE, BL, and HR (average, max, and percentage of time ≥80% HRmax) were highest in the knockout bouts compared with poules (8.5 ± 1.3 vs. 5.7 ± 1.3, 3.6 ± 1.0 vs. 3.1 ± 1.4 mmol·L, 171 ± 5 vs. 168 ± 8 b·min, 195 ± 7 vs. 192 ± 7 b·min, 74 vs. 68%); however, only significant (p ≤ 0.05) for RPE. Countermovement jump height, albeit nonsignificantly (p > 0.05), increased throughout competition and dropped thereafter. Although responses of C, AA, and IgA showed a tendency to increase during competition and drop thereafter (T and T:C doing the opposite), no significant differences were noted for any analyte. Results suggest that fencing is a high-intensity anaerobic sport, relying on alactic energy sources. However, some bouts evoke BL values of ≥4 mmol·L and thus derive energy from anaerobic glycolysis. High HRs appear possible on account of ample within- and between-bout rest. The small competition load associated with fencing competitions may explain the nonsignificant findings noticed.

Placental syndecan-1 and sulphated glycosaminoglycans are decreased in preeclampsia.

Glycosaminoglycans are found in extracellular matrix and on the cell surface in the form of proteoglycans. There is evidence that these molecules regulate biological processes, including cell survival, migration and angiogenesis. Preeclampsia is a common pregnancy disorder associated with insufficient placental development. This study aimed to determine the concentrations of glycosaminoglycans and the proteoglycan syndecan-1 within villous trophoblast and to investigate changes associated with preeclampsia. Seventy-five placental samples collected from third trimester singleton pregnancies were divided into term placentas following labour onset, gestational age-matched placentas prior to labour onset and preterm placentas. Preterm placentas were divided into three gestational age-matched groups, spontaneous preterm labour, preterm premature rupture of membranes (PPROM) and preterm preeclampsia. Sulphated glycosaminoglycan (sGAG) concentrations in placental extracts were quantified using a modified 1,9-dimethylmethylene blue assay. Syndecan-1 expression was localised using immunohistochemistry and concentrations in placental extracts determined by immunoassay. Preterm placentas had significantly lower sGAG concentrations compared to term tissues and concentrations were significantly lower in preeclampsia compared to spontaneous preterm labour (medians 5.80 and 10.0 µg/mg protein respectively, P<0.05). Syndecan-1 expression was localised to syncytiotrophoblast and median concentrations were lower in preeclampsia compared to PPROM material (preeclampsia median = 41.7, PPROM 74.4 ng/mg tissue) but not significantly different to concentrations in spontaneous preterm labour. Multivariate analysis revealed that decreased sGAG and syndecan-1 in preeclampsia were independent of labour, gestational age and birthweight centile. These findings may provide insights into a role for these molecules in the pathophysiology of preeclampsia.

Disaccharide compositional analysis of chondroitin sulphate using WAX HILIC-MS with pre-column procainamide labelling; application to the placenta in pre-eclampsia.

Chondroitin sulphate (CS) and dermatan sulphate are negatively charged linear heteropolysaccharides. These glycosaminoglycans (GAG) are involved in cellular signalling via binding to growth factors. CS is expressed in a range of tissue and biological fluids and is highly expressed in the placenta. There is evidence that decorin; a CS proteoglycan is significantly decreased in pre-eclampsia and fetal growth restriction. It is considered that GAG chain composition may influence cellular processes that are altered in pre-eclampsia. The goal of the present study was to develop an LC-MS method with precolumn procainamide labelling for the disaccharide compositional analysis of CS. The method was used to investigate whether the disaccharide composition of placenta-extracted CS is altered in pre-eclampsia. The study revealed differential disaccharide compositions of placental chondroitin sulphate between pre-eclampsia and other pregnancy conditions. This suggests that the method may have diagnostic potential for pregnancy disorders. Furthermore, the findings suggest that CS sulphation might play a significant role in maternal labour.

Differential proteolysis of insulin-like growth factor binding protein-1 (IGFBP-1) in pregnancy.

The insulin-like growth factors and their binding proteins are important for placental and foetal growth. In this study, we have investigated the presence of proteolytic activity directed against insulin-like growth factor binding protein-1 (IGFBP-1) in pregnancy. In addition, the effect of protease activity on IGFBP-1 immunoreactivity and IGF binding was characterised. 125I-IGFBP-1 was incubated with maternal and foetal serum, amniotic fluid and placental extracts. Breakdown of 125I-IGFBP-1 was determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography. The size distribution of endogenous IGFBP-1 was determined by Western immunoblotting. Protease inhibitor studies characterised the proteolytic activity, and Western ligand blotting with 125I-IGF-I was used to determine IGF binding capacity of proteolysed IGFBP-1. Amniotic fluid samples collected after labour onset contained proteolytic activity that generated 12- and 19-kDa IGFBP-1 fragments that did not bind to 125I-IGF-I. This activity was not detected in amniotic fluid collected prior to labour onset or in other tissues. Activity was blocked by aprotinin, leupeptin, phenyl methyl sulphonyl fluoride, and Kunitz soybean trypsin inhibitor but not by ethylene diamine tetraacetic acid or pepstatin. Incubation of IGFBP-1 with trypsin generated fragments of a similar size to the amniotic fluid protease. In conclusion, we have demonstrated the presence in vivo of a trypsin-like proteolytic activity that alters the IGF-binding function of IGFBP-1 in pregnancy.

Effect of hot water immersion on acute physiological responses following resistance exercise.

Purpose: Hot water immersion (HWI) is a strategy theorised to enhance exercise recovery. However, the acute physiological responses to HWI following resistance exercise are yet to be determined. Methods: The effect of HWI on intramuscular temperature (IMT), muscle function, muscle soreness and blood markers of muscle cell disruption and inflammatory processes after resistance exercise was assessed. Sixteen resistance trained males performed resistance exercise, followed by either 10 min HWI at 40°C or 10 min passive recovery (PAS). Results: Post-intervention, the increase in IMT at all depths was greater for HWI compared to PAS, however this difference had disappeared by 1 h post at depths of 1 and 2 cm, and by 2 h post at a depth of 3 cm. There were no differences between groups for muscle function, muscle soreness or any blood markers. Conclusion: These results suggest that HWI is a viable means of heat therapy to support a greater IMT following resistance exercise. Recovery of muscle function and muscle soreness is independent of acute changes in IMT associated with HWI.

A combination of cherry juice and cold water immersion does not enhance marathon recovery compared to either treatment in isolation: A randomized placebo-controlled trial.

Purpose: Cherry juice (CJ) and cold water immersion (CWI) are both effective recovery strategies following strenuous endurance exercise. However, athletes routinely combine recovery interventions and less is known about the impact of a combined CJ and CWI protocol. Therefore, this study investigated the effects of combining CWI and CJ (a "cocktail" (CT)) on inflammation and muscle damage following a marathon. Methods: A total 39 endurance trained males were randomly assigned to a placebo (PL), CWI, CJ, or CT group before completing a trail marathon run. Muscle damage (creatine kinase (CK)), muscle function (maximal voluntary isometric contraction (MVIC)), and inflammation (interleukin-6 (IL-6); C-reactive protein (CRP)) were measured at baseline, immediately after marathon (only IL-6), 24 h, and 48 h after marathon. Results: There were no statistically significant differences between groups and no group × time interaction effects for any of the dependent variables. Confidence intervals (CI) illustrated that CT had unclear effects on inflammation (IL-6; CRP) and MVIC, but may have increased CK to a greater extent than PL and CJ conditions. Conclusion: There is no evidence of an additive effect of CJ and CWI when the treatments are used in conjunction with each other. On the contrary, combining CJ and CWI may result in slightly increased circulating CK.

Reciprocal imprinting of human GRB10 in placental trophoblast and brain: evolutionary conservation of reversed allelic expression.

Genomic imprinting may have evolved not only to regulate fetal growth and development, but also behaviour. The mouse Grb10 gene provides a remarkable model to explore this idea because it shows paternal expression in brain, whereas in the placenta and most other embryonic tissues, expression is from the maternal allele. To assess the biological relevance of this reciprocal pattern of imprinting, we explored its conservation in humans. As in mice, we find the human GRB10 gene to be paternally expressed in brain. Maternal allele-specific expression is conserved only in the placental villous trophoblasts, an essential part of the placenta involved in nutrient transfer. All other fetal tissues tested showed equal expression from both alleles. These data suggest that the maternal GRB10 expression in placenta is evolutionarily important, presumably in the control of fetal growth. As in the mouse, the maternal transcripts originate from several kilobases upstream of the imprinting control region (ICR) of the domain, from a promoter region at which we find no allelic chromatin differences. The brain-specific paternal expression from the ICR shows mechanistic similarities with the mouse as well. This conserved CpG island is DNA-methylated on the maternal allele and is marked on the paternal allele by developmentally regulated bivalent chromatin, with the presence of both H3 lysine-4 and H3 lysine-27 methylation. The strong conservation of the opposite allelic expression in placenta versus brain supports the hypothesis that GRB10 imprinting evolved to mediate diverse roles in mammalian growth and behaviour.

The effect of milk on recovery from repeat-sprint cycling in female team-sport athletes.

The consumption of milk following eccentric exercise attenuates the effects of muscle damage in team-sport athletes. However, participation in team sport involves both concentric-eccentric loading and metabolic stress. Therefore, the aim of this study was to investigate the effects of postexercise milk consumption on recovery from a cycling protocol designed to simulate the metabolic demands of team sport. Ten female team-sport athletes participated in a randomised crossover investigation. Upon completion of the protocol participants consumed 500 mL of milk (MILK) or 500 mL of an energy-matched carbohydrate (CHO) drink. Muscle function (peak torque, rate of force development, countermovement jump, 20-m sprint), muscle soreness and tiredness, serum creatine kinase, high-sensitivity C-reactive protein, and measures of oxidative stress (protein carbonyls and reduced glutathione/oxidized glutathione (GSH/GSSG) ratio) were determined at pre-exercise and 24 h, 48 h, and 72 h postexercise. MILK had a possible beneficial effect in attenuating losses in peak torque (180°/s) from baseline to 24 h (3.2% ± 7.8% vs. -6.2% ± 7.5%, MILK vs. CHO) and a possible beneficial effect in minimising soreness (baseline-48 h; baseline-72 h) and tiredness (baseline-24 h; baseline-72 h). There was no change in oxidative stress following the exercise protocol, though a likely benefit of milk was observed for GSH/GSSG ratio at baseline-24 h (0.369 ×/÷ 1.89, 1.103 ×/÷ 3.96, MILK vs. CHO). MILK had an unclear effect on all other variables. Consumption of 500 mL of milk after repeat sprint cycling had little to no benefit in minimising losses in peak torque or minimising increases in soreness and tiredness and had no effect on serum markers of muscle damage and inflammation.

Insulin-like growth factor-II and heparin are anti-apoptotic survival factors in human villous cytotrophoblast.

OBJECTIVE: This study aimed to determine the effects of insulin-like growth factors (IGF-I and IGF-II), heparin, aspirin and vitamin C on the proliferation and apoptosis of human villous cytotrophoblast from first trimester and term placentae. STUDY DESIGN: Villous cytotrophoblast cells were isolated from uncomplicated first trimester (n=12) and term placental tissues (n=12) using negative immunoselection with an antibody to HLA class I antigens. Cells were incubated with IGF-I, IGF-II, heparin, aspirin and vitamin C either alone, or in combination with either TNF-α/IFN-γ or staurosporine. Proliferation was determined by measurement of Ki67 expression using immunocytochemistry. Trophoblast apoptosis was determined by TUNEL staining. Finally RT-PCR was carried out to identify IGF-binding insulin receptor isoforms. Data were expressed as means±SEM. One way analysis of variance (ANOVA) with Bonferroni correction was used to determine if differences between groups were statistically significant. RESULTS: Following negative immunoselection >98% of cells were positively stained for cytokeratin 7, a marker for cytotrophoblasts, and <1% were vimentin positive. First trimester and term trophoblasts underwent spontaneous apoptosis which was inhibited by approximately 50% in the presence of IGF-II or heparin. Apoptosis was significantly increased following incubation with a combination of TNF-α and IFN-γ or staurosporine. Apoptosis was decreased to basal levels following coincubation with IGF-II or heparin. Incubation with IGFs or heparin resulted in a small, but significant increase in Ki67 expression. Insulin receptor isoform A, which binds IGF-II with high affinity, was present in all trophoblast samples tested. CONCLUSION: These results suggest that heparin and IGF-II, but not IGF-I are important regulators of villous cytotrophoblast survival in early and late pregnancy.

Heparin prevents programmed cell death in human trophoblast.

Heparin is used clinically for the prevention of pregnancy complications associated with prothrombotic disorders, especially antiphospholipid antibody syndrome. Recent studies have suggested that heparin may exert direct effects on placental trophoblast, independently of its anticoagulant activity. We now demonstrate that heparin abrogates apoptosis of primary first trimester villous trophoblast in response to treatment with the pro-inflammatory cytokines interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha. This multifunctional glycosaminoglycan also inhibited apoptosis induced by other agents, including staurosporin, broad-spectrum kinase inhibitor and thrombin. Furthermore, heparin attenuated caspase-3 activity, a hallmark of apoptosis, in human first trimester villous and extravillous trophoblast cell lines treated with peptidoglycan, a Toll-like receptor-2 agonist isolated from Staphylococcus aureus. The ability of heparin to antagonize cell death induced by such diverse apoptotic signals suggested that it acts as a survival factor for human trophoblast. We demonstrate that heparin, like epidermal growth factor (EGF) and heparin-binding EGF (HB-EGF), elicits phosphorylation of the EGF receptor and activation of the phosphatidyl inositol 3-kinase (PI3K)-, the extracellular signal-related kinase 1/2 (ERK1/2)- and the c-Jun NH2 terminal kinase (JNK)-signal transduction pathways in primary villous trophoblast. In summary, we have demonstrated that heparin activates multiple anti-apoptotic pathways in human trophoblast. Our results suggest that heparin may be useful in the management of at-risk patients, even in the absence of an identifiable thrombophilic disorder.