Tackling Kinesiophobia in Chronic Shoulder Pain: A Case Report on the Combined Effect of Pain Education and Whole-Body Cryostimulation.

BACKGROUND: Chronic shoulder pain may cause significant functional disability and reduced psychosocial well-being. Detailed Case Description: In this case, we propose the use of pain neuroscience education and whole-body cryostimulation (WBC) to treat a 64-year-old woman with severe functional limitations and chronic right shoulder pain. The aim was to overcome kinesiophobia and improve her motor function, autonomy, and quality of life. Functional and clinical assessments were conducted at admission, discharge, and at a one-month follow-up via phone call. The patient's global health, shoulder function, and quality of life showed improvement during hospitalisation and were maintained after one month. DISCUSSION: Pain education is crucial in managing chronic shoulder pain, especially in addressing kinesiophobia and promoting positive patient outcomes. In this context, WBC was used as a supplementary treatment to traditional pain relief and exercise tolerance therapies. This can help individuals to participate more actively in their rehabilitation process, ultimately promoting functional recovery and an improved quality of life. CONCLUSION: The combination of cryostimulation, tailored physical exercises, pain education, manual therapy, and psychological support created a synergistic effect that addressed both the physical and psychological aspects of pain and kinesiophobia.

Whole-Body Cryostimulation in Multiple Sclerosis: A Scoping Review.

Multiple sclerosis (MS) is the most common cause of non-traumatic long-term disability in young adults. Whole-body cryostimulation (WBC) is a cold-based physical therapy known to induce physiological exercise-mimicking changes in the cardiovascular, neuromuscular, immune, and endocrine systems and to influence functional and psychological parameters by exposing the human body to cryogenic temperatures (≤-110 °C) for 2-3 min. The purpose of this scoping review is to present an overall view on the potential role of WBC as an adjuvant therapy in the treatment of MS. PubMed, ScienceDirect, Embase, and Web of Science were searched up to 30 November 2023, and a total of 13 articles were included. WBC may have beneficial antioxidant effects as a short-term adjuvant treatment in MS. There were no significant changes in antioxidant enzymes, nitric oxide levels, metalloproteinase levels, blood counts, rheology, and biochemistry. WBC can lead to a reduction in fatigue and an improvement in functional status, with a significant effect on both mental and physical well-being. There were no reported adverse effects. The results suggest that WBC may complement therapeutic options for patients with MS, as the effects of cryogenic cold stimulation have been shown to activate antioxidant processes and improve functional status, mood, anxiety, and fatigue.

Whole Body Cryostimulation: A New Adjuvant Treatment in Central Sensitization Syndromes? An Expert Opinion.

Central sensitisation is defined as a multifactorial etiopathogenetic condition involving an increase in the reactivity of nociceptive neurons and alterations in pain transmission and perception in the central nervous system. Patients may present with widespread chronic pain, fatigue, sleep disturbance, dizziness, psychological (e.g., depression, anxiety, and anger) and social impairment. Pain can be spontaneous in onset and persistence, characterised by an exaggerated response and spread beyond the site of origin, and sometimes triggered by a non-painful stimulus. Whole-body cryostimulation (WBC) could be an adjuvant therapy in the management of this type of pain because of its global anti-inflammatory effect, changes in cytokines and hormone secretion, reduction in nerve conduction velocity, autonomic modulation, and release of neurotransmitters involved in the pain pathway. In several conditions (e.g., fibromyalgia, rheumatoid arthritis, and chronic musculoskeletal pain), WBC affects physical performance, pain perception, and psychological aspects. Given its multiple targets and effects at different organs and levels, WBC appears to be a versatile adjuvant treatment for a wide range of conditions of rehabilitation interest. Further research is needed to fully understand the mechanisms of analgesic effect and potential actions on pain pathways, as well as to study long-term effects and potential uses in other chronic pain conditions.

Whole-Body Cryostimulation as an Adjunctive Treatment for Neurophysiologic Tinnitus and Associated Disorders: Preliminary Evidence from a Case Study.

BACKGROUND: Tinnitus, which is often associated with reduced quality of life, depression, and sleep disturbances, lacks a definitive treatment targeting its pathophysiological mechanism. Inflammatory markers like TNF-α have been linked to tinnitus, thereby underlining the necessity for innovative therapies. This case study investigates the potential benefits of a multi-approach rehabilitation intervention involving whole-body cryostimulation (WBC) for a 47-year-old male suffering from chronic neurophysiologic tinnitus, who had underwent various unsuccessful treatments from 2005. METHODS: the patient underwent a personalized, multidisciplinary rehabilitation intervention covering diet, pharmacotherapy, physiotherapy and physical activity classes tailored to the patient's needs and capacities, repetitive transcranial magnetic stimulation (rTMS), and whole-body cryostimulation (WBC). RESULTS: The adjunctive WBC intervention resulted in a significant progressive improvement in tinnitus severity (tinnitus handicap inventory Δ% = -46.3%, VAS tinnitus score Δ% = -40%). Additional positive outcomes were noted in sleep quality (PSQI Δ% = -41.67%), emotional wellbeing (BDI Δ% = -41.2%), and quality of life (SF-36, WHO-5 Δ% = +16.5). CONCLUSIONS: This study supports the existing literature suggesting the potential of WBC as an adjunct in a multi-approach intervention in ameliorating tinnitus severity and tinnitus-associated disorders. However, randomized controlled trials in larger populations, which specifically consider WBC's effects on tinnitus, are necessary to confirm these findings and to explore the mechanisms that underlie the observed improvements.

Whole-Body Cryostimulation in Post-COVID Rehabilitation for Patients with Obesity: A Multidisciplinary Feasibility Study.

BACKGROUND: A post-COVID condition can reduce activity and quality of life, resulting in a significant socioeconomic and health burden. Understanding its impact on patients' health is important for the development of personalized rehabilitation interventions. An independent association between obesity and post-COVID condition was found because of complications and comorbidities. METHODS: Sixteen patients with obesity and post-COVID symptoms (i.e., dyspnea, pain, poor sleep quality, muscle fatigue), admitted to the Istituto Auxologico Italiano, Piancavallo (VB), Italy, were recruited for a four-week rehabilitation program including conventional exercise therapy, nutritional intervention, psychological support and whole-body cryostimulation (WBC). RESULTS: All participants attended all sessions of the program. Anthropometric data showed statistically significant changes in weight, waist circumference and body mass index. Biochemical analyses showed significant reductions in lipid and inflammatory profiles. There was a significant improvement in physical performance, reduction in pain and improvement in psychological well-being. CONCLUSION: A multidisciplinary rehabilitation protocol including WBC, designed for patients with obesity and a post-COVID condition, is safe and feasible. The overall improvements demonstrate that multidisciplinary rehabilitation was effective on post COVID patients and suggest that the use of WBC is safe and could play a role as a booster in rehabilitation programs.

Effects of Whole-Body Cryostimulation on Pain Management and Disease Activity in Active Rheumatic Polymyalgia: A Case-Report.

Polymyalgia rheumatica (PMR) is the most common inflammatory rheumatic disease among people over 50 and occurs with symptoms such as musculoskeletal pain and stiffness in the neck, shoulders, and hips. To date, corticosteroids represent the cornerstone of PMR treatment. However, it is well known that their prolonged use is associated with several adverse effects, making it crucial to find therapeutic alternatives. The purpose of this case report was to describe the effectiveness of 10 whole-body cryostimulation (WBC) sessions on a 74-year-old woman suffering from PMR. An improvement in disease impact, fatigue, pain, quality of sleep, and total physical activity was observed after WBC. Moreover, the patient reduced her daily drug intake by 67% following WBC treatments. Given the increasing prevalence of PMR and considering the side effects that drug treatments can lead to, WBC could represent a valuable adjuvant and well-tolerated alternative for treating PMR.

Whole-Body Cryostimulation, a Complementary Treatment for Phantom Limb Syndrome: Preliminary Evidence from a Case Study.

Phantom limb pain (PLP) is a challenging condition affecting a significant proportion of amputees. In this article, we describe the case of a 54-year-old Paralympic athlete with phantom limb syndrome following right leg amputation and widespread sports-related enthesitic pain who underwent a whole-body cryostimulation (WBC) cycle, an emerging treatment known for its rapid pain-relieving and anti-inflammatory effects. Assessments were conducted before and after a 10-session WBC cycle, including pain and quality of life assessment and use of medications. A substantial reduction in enthesitic pain, PLP intensity, paresthesia, and tingling related to atmospheric events and improved function and quality of life were reported after the WBC cycle and lasted for two weeks. One month after WBC, the enthesitic pain following sports activity and PLP gradually returned, but with lesser intensity. Similarly, the stump's sensitivity to atmospheric changes returned, but with lower frequency. Pain at night remained lower than before WBC, with significantly improved quality of sleep. This case study suggests that WBC could be a valuable adjuvant treatment for alleviating PLP. Controlled studies are warranted to validate the findings of this case report and elucidate the mechanisms underlying the positive effects of WBC in this condition.

Whole-Body Cryostimulation in Functional Neurological Disorders: A Case Report.

Functional neurological disorders (FNDs) are complex disabling conditions requiring a multiple rehabilitation intervention. Here, we propose a new use of whole-body cryostimulation (WBC) that was implemented in a multidisciplinary rehabilitation programme in a wheelchair-ridden woman diagnosed with FND and other comorbidities. WBC is a promising adjuvant treatment in various conditions of rehabilitation interest, mainly because of its wide range of rapid effects, from anti-inflammatory to pain and autonomic modulating effects. The 4-week program included physiotherapy, nutritional intervention, psychological support, and WBC (-110 °C for 2 min). Questionnaires to assess disease impact, pain level, fatigue and sleep quality were administered. At discharge, improvements in body composition, haematological biomarkers, physical performance, and questionnaire scores were observed. The patient was able to walk independently with a walker for medium distances and reported unprecedented improvements, particularly in functional parameters and questionnaire scores. Although the extent to which WBC per se contributed to the measured improvements cannot be ascertained, subjective reports and our clinical observations indicate that WBC, the only intervention not previously experienced by the patient, acted as a booster for the rehabilitation interventions. Further research will be necessary to rule out any possible placebo effect and to confirm the effects of WBC on FND.

Catastrophizing, Kinesiophobia, and Acceptance as Mediators of the Relationship Between Perceived Pain Severity, Self-Reported and Performance-Based Physical Function in Women with Fibromyalgia and Obesity.

Purpose: Individuals with fibromyalgia and obesity experience significant impairment in physical functioning. Pain catastrophizing, kinesiophobia, and pain acceptance have all been identified as important factors associated with the level of disability. The objective of this study was to evaluate the role of pain catastrophizing, kinesiophobia, and pain acceptance as mediators of the association between perceived pain severity and physical functioning in individuals with fibromyalgia and obesity. Patients and Methods: In this cross-sectional study, 165 women with fibromyalgia and obesity completed self-report questionnaires of perceived pain severity (ie, Numeric Pain Rating Scale), pain catastrophizing (ie, Pain Catastrophizing Scale), kinesiophobia (ie Tampa Scale of Kinesiophobia), pain acceptance (ie, Chronic Pain Acceptance Questionnaire), and perceived physical functioning (ie, Physical Functioning subscale of the Fibromyalgia Impact Questionnaire). In addition, a performance-based test (ie, 6-minute walking test) was conducted to assess objective physical functioning. Two multiple mediation analyses were performed. Results: Pain acceptance and kinesiophobia mediated the relationship between pain severity and self-reported physical functioning. Pain catastrophizing and kinesiophobia mediated the relationship between pain severity and performance-based functioning. Conclusion: Pain acceptance, kinesiophobia, and pain catastrophizing should be addressed in rehabilitative intervention to improve physical functioning. Interestingly, the subjective and objective aspects of physical functioning are influenced by different factors. Therefore, interventions for women with fibromyalgia and obesity should focus on factors related to both subjective and performance-based physical functioning.

WHOLE-BODY CRYOSTIMULATION: A REHABILITATION BOOSTER.

A growing body of work suggests that whole-body cryostimulation (WBC) could play a role as a promising adjuvant therapy in various conditions of rehabilitation interest. In fact, WBC is currently being used to relieve symptoms in rheumatoid arthritis, fibromyalgia, ankylosing spondylitis, depression and anxiety, multiple sclerosis, sleep disturbances, muscle soreness after strenuous physical exercise, post-Covid syndrome and obesity. WBC is not only a symptomatic physical therapy but rather represents an "adaptation therapy" because of the repeated shock-like cryogenic cold stimulus over the entire body surface that induces reactions in the autonomic, endocrine, circulatory, neuromuscular and immunological systems, resulting in an adaptation that contributes to the restoration of the homeostatic state. Therefore, based on the existing evidence, WBC can be described as follows:a "training method" for the autonomic nervous system;a novel anti-inflammatory and antioxidant treatment;a treatment with beneficial effects on body composition and adipose tissue. In our opinion, the powerful effects of thermal stress on the physiological responses of the human body present unique features that could potentially be exploited to boost rehabilitation outcomes in various conditions. Therefore, we believe it is important to highlight the potential use of WBC for medical use and emphasize its relevance in the field of rehabilitation with the aim of stimulating scientific studies on the efficacy of WBC as an adjuvant treatment in various conditions of rehabilitation interest.

Telomere Length and Mitochondrial DNA Copy Number Variations in Patients with Obesity: Effect of Diet-Induced Weight Loss-A Pilot Study.

Background: Telomere length (TL) and mitochondrial DNA (mtDNA) copy number shifts are linked to metabolic abnormalities, and possible modifications by diet-induced weight loss are poorly explored. We investigated the variations before (T0) and after a 1-year (T12) lifestyle intervention (diet + physical activity) in a group of outpatients with obesity. Methods: Patients aged 25−70 years with BMI ≥ 30 kg/m2 were enrolled. Clinical and biochemical assessments (including a blood sample for TL, mtDNA copy number and total antioxidant capacity, and TAC determinations) were performed at T0 and T12. Results: The change in TL and the mtDNA copy number was heterogeneous and not significantly different at T12. Patients were then divided by baseline TL values into lower than median TL (L-TL) and higher than median TL (H-TL) groups. The two groups did not differ at baseline for anthropometric, clinical, and laboratory characteristics. At T12, the L-TL group when compared to H-TL showed TL elongation (respectively, +0.57 ± 1.23 vs. −2.15 ± 1.13 kbp, p = 0.04), higher mtDNA copy number (+111.5 ± 478.5 vs. −2314.8 ± 724.2, respectively, p < 0.001), greater weight loss (−8.1 ± 2.7 vs. −6.1 ± 4.6 Kg, respectively, p = 0.03), fat mass reduction (−1.42 ± 1.3 vs. −1.22 ± 1.5%, respectively, p = 0.04), and increased fat-free mass (+57.8 ± 6.5 vs. +54.9 ± 5.3%, respectively, p = 0.04) and TAC levels (+58.5 ± 18.6 vs. +36.4 ± 24.1 µM/L, respectively, p = 0.04). Conclusions: TL and the mtDNA copy number significantly increased in patients with obesity and with lower baseline TL values after a 1-year lifestyle intervention. Larger longitudinal studies are needed to confirm the results of this pilot study.

Is Whole-Body Cryostimulation an Effective Add-On Treatment in Individuals with Fibromyalgia and Obesity? A Randomized Controlled Clinical Trial.

Pain severity, depression, and sleep disturbances are key targets for FM rehabilitation. Recent evidence suggests that whole-body cryostimulation (WBC) might be an effective add-on treatment in the management of FM. The purpose of this study was to evaluate the effects of an add-on WBC intervention to a multidisciplinary rehabilitation program on pain intensity, depressive symptoms, disease impact, sleep quality, and performance-based physical functioning in a sample of FM patients with obesity. We performed a randomized controlled trial with 43 patients with FM and obesity undergoing a multidisciplinary rehabilitation program with and without the addition of ten 2-min WBC sessions at -110 °C over two weeks. According to our results, the implementation of ten sessions of WBC over two weeks produced additional benefits. Indeed, both groups reported positive changes after the rehabilitation; however, the group that underwent WBC intervention had greater improvements in the severity of pain, depressive symptoms, disease impact, and quality of sleep. On the contrary, with respect to performance-based physical functioning, we found no significant between-group differences. Our findings suggest that WBC could be a promising add-on treatment to improve key aspects of FM, such as pain, depressive symptoms, disease impact and poor sleep quality.

Whole-body cryostimulation in obesity. A scoping review.

Currently available treatments for the management of obesity struggle to provide clinically significant weight loss and reduction of the chronic low-grade inflammatory state in order to reduce obesity-related complications. This scoping review aims to provide an up-to-date picture of the therapeutic effects of Whole-Body Cryostimulation (WBC) in patients with obesity and evidence-based indications for its complementary use in the treatment of obesity. We searched the literature until the end of August 2021, retrieving 8 eligible studies out of 856, all evaluated for their methodological quality using the Downs and Black checklist. Overall, the limited data presented in this review article seem to support the efficacy of WBC as an adjuvant treatment in obesity. The cryogenic stimulus has important anti-inflammatory/antioxidant effects and its effectiveness is directly related to the individual percentage of fat mass and initial fitness capacity, mimicking an exercise-induced effect. Based on the limited results gathered, WBC emerges as a promising adjuvant therapy to reduce systemic inflammation, oxidative stress, abdominal obesity, and body mass. However, the data presented in this review article fail to reach definitive conclusions with regards to the efficacy of WBC in the treatment of obesity. Application of WBC protocols yields the potential to widen the therapeutic armor for the treatment of obesity and obesity-related disorders but larger, high-quality studies are still needed.

Differential effects of noise exposure between substrains of CBA mice.

Noise trauma involves a plethora of mechanisms including reactive oxygen species, apoptosis, tissue damage, and inflammation. Recently, circadian mechanisms were also found to contribute to the vulnerability to noise trauma in mice, with greater damage occurring during their active phase (nighttime), when compared to similar noise exposures during their inactive phase (daytime). These effects seem to be regulated by mechanisms involving Bdnf responses to noise trauma and circulating levels of corticosterone (CORT). However, recent studies using different noise paradigms show contradicting results and it remains unclear how universal these findings are. Here we show that these findings differ even between substrains of mice and are restricted to a narrow window of noise intensity. We found that CBA/Sca mice exposed to 103 dB SPL display differential day/night noise sensitivity as measured by auditory brainstem responses (ABRs), but not at 100 (where full recovery is observed in day or night exposed mice) or 105 dB SPL (where permanent damage is found in both groups). In contrast, neither CBA/CaJ or CBA/JRj displayed such differences in day/night noise sensitivity, whatever noise intensity used. These effects appeared to be independent from outer hair cell function, as distortion product otoacoustic emissions appeared equally affected by day or night noise exposure, in all strains and in all noise conditions. Minor differences in ribbon counts or synaptic pairing were found in CBA/Sca mice, which were inconsistent with ABR wave 1 amplitude changes. Interestingly, CORT levels peaked in CBA/Sca mice at the onset of darkness at zeitgeber time 12 reaching levels of 43.8 ng/ml, while in the CBA/CaJ and the CBA/JRj, levels were 11.9 and 15.6 ng/ml respectively and peaking 4 h earlier (zeitgeber time 8). These findings were consistent with higher period of daily rhythm in CBA/Sca mice when measured in complete darkness using running wheels (23.7 h), than in CBA/CaJ (23.45 h) or CBA/JRj (23.13 h). In conclusion, our study suggests that the differential vulnerability to noise trauma between inactive and active phase is not universal and is as sensitive as substrain differences that might be governed by the circadian amplitude of the circulating CORT profiles.

Super-resolution microscopy reveals that Na+/K+-ATPase signaling protects against glucose-induced apoptosis by deactivating Bad.

Activation of the apoptotic pathway is a major cause of progressive loss of function in chronic diseases such as neurodegenerative and diabetic kidney diseases. There is an unmet need for an anti-apoptotic drug that acts in the early stage of the apoptotic process. The multifunctional protein Na+,K+-ATPase has, in addition to its role as a transporter, a signaling function that is activated by its ligand, the cardiotonic steroid ouabain. Several lines of evidence suggest that sub-saturating concentrations of ouabain protect against apoptosis of renal epithelial cells, a common complication and major cause of death in diabetic patients. Here, we induced apoptosis in primary rat renal epithelial cells by exposing them to an elevated glucose concentration (20 mM) and visualized the early steps in the apoptotic process using super-resolution microscopy. Treatment with 10 nM ouabain interfered with the onset of the apoptotic process by inhibiting the activation of the BH3-only protein Bad and its translocation to mitochondria. This occurred before the pro-apoptotic protein Bax had been recruited to mitochondria. Two ouabain regulated and Akt activating Ca2+/calmodulin-dependent kinases were found to play an essential role in the ouabain anti-apoptotic effect. Our results set the stage for further exploration of ouabain as an anti-apoptotic drug in diabetic kidney disease as well as in other chronic diseases associated with excessive apoptosis.

Impact of noise exposure on the circadian clock in the auditory system.

Circadian rhythms control the timing of all bodily functions, and misalignment in the rhythms can cause various diseases. Moreover, circadian rhythms are highly conserved and are regulated by a transcriptional-translational feedback loop of circadian genes that has a periodicity of approximately 24 h. The cochlea and the inferior colliculus (IC) have been shown to possess an autonomous and self-sustained circadian system as demonstrated by recording, in real time, the bioluminescence from PERIOD2::LUCIFERASE (PER2::LUC) mice. The cochlea and IC both express the core clock genes, Per1, Per2, Bmal1, and Rev-Erbα, where RNA abundance is rhythmically distributed with a 24 h cycle. Noise exposure alters clock gene expression in the cochlea and the IC after noise stimulation, although in different ways. These findings highlight the importance of circadian responses in the cochlea and the IC and emphasize the importance of circadian mechanisms for understanding the differences in central and peripheral auditory function and the subsequent molecular changes that occur after daytime (inactive phase) or nighttime (active phase) noise trauma.

Ouabain-regulated phosphoproteome reveals molecular mechanisms for Na+, K+-ATPase control of cell adhesion, proliferation, and survival.

The ion pump Na+, K+-ATPase (NKA) is a receptor for the cardiotonic steroid ouabain. Subsaturating concentration of ouabain triggers intracellular calcium oscillations, stimulates cell proliferation and adhesion, and protects from apoptosis. However, it is controversial whether ouabain-bound NKA is considered a signal transducer. To address this question, we performed a global analysis of protein phosphorylation in COS-7 cells, identifying 2580 regulated phosphorylation events on 1242 proteins upon 10- and 20-min treatment with ouabain. Regulated phosphorylated proteins include the inositol triphosphate receptor and stromal interaction molecule, which are essential for initiating calcium oscillations. Hierarchical clustering revealed that ouabain triggers a structured phosphorylation response that occurs in a well-defined, time-dependent manner and affects specific cellular processes, including cell proliferation and cell-cell junctions. We additionally identify regulation of the phosphorylation of several calcium and calmodulin-dependent protein kinases (CAMKs), including 2 sites of CAMK type II-γ (CAMK2G), a protein known to regulate apoptosis. To verify the significance of this result, CAMK2G was knocked down in primary kidney cells. CAMK2G knockdown impaired ouabain-dependent protection from apoptosis upon treatment with high glucose or serum deprivation. In conclusion, we establish NKA as the coordinator of a broad, tightly regulated phosphorylation response in cells and define CAMK2G as a downstream effector of NKA.-Panizza, E., Zhang, L., Fontana, J. M., Hamada, K., Svensson, D., Akkuratov, E. E., Scott, L., Mikoshiba, K., Brismar, H., Lehtiö, J., Aperia, A. Ouabain-regulated phosphoproteome reveals molecular mechanisms for Na+, K+-ATPase control of cell adhesion, proliferation, and survival.

Circadian integration of inflammation and glucocorticoid actions: Implications for the cochlea.

Auditory function has been shown to be influenced by the circadian system. Increasing evidence point towards the regulation of inflammation and glucocorticoid actions by circadian rhythms in the cochlea. Yet, how these three systems (circadian, immune and endocrine) converge to control auditory function remains to be established. Here we review the knowledge on immune and glucocorticoid actions, and how they interact with the circadian and the auditory system, with a particular emphasis on cochlear responses to noise trauma. We propose a multimodal approach to understand the mechanisms of noise-induced hearing loss by integrating the circadian, immune and endocrine systems into the bearings of the cochlea. Considering the well-established positive impact of chronotherapeutic approaches in the treatment of cardiovascular, asthma and cancer, an increased knowledge on the mechanisms where circadian, immune and glucocorticoids meet in the cochlea may improve current treatments against hearing disorders.

Spontaneous calcium activity in metanephric mesenchymal cells regulates branching morphogenesis in the embryonic kidney.

The central role of calcium signaling during development of early vertebrates is well documented, but little is known about its role in mammalian embryogenesis. We have used immunofluorescence and time-lapse calcium imaging of cultured explanted embryonic rat kidneys to study the role of calcium signaling for branching morphogenesis. In mesenchymal cells, we recorded spontaneous calcium activity that was characterized by irregular calcium transients. The calcium signals were dependent on release of calcium from intracellular stores in the endoplasmic reticulum. Down-regulation of the calcium activity, both by blocking the sarco-endoplasmic reticulum Ca2+-ATPase and by chelating cytosolic calcium, resulted in retardation of branching morphogenesis and a reduced formation of primitive nephrons but had no effect on cell proliferation. We propose that spontaneous calcium activity contributes with a stochastic factor to the self-organizing process that controls branching morphogenesis, a major determinant of the ultimate number of nephrons in the kidney.-Fontana, J. M., Khodus, G. R., Unnersjö-Jess, D., Blom, H., Aperia, A., Brismar, H. Spontaneous calcium activity in metanephric mesenchymal cells regulates branching morphogenesis in the embryonic kidney.

Transport and release of colloidal 3-mercaptopropionic acid-coated CdSe-CdS/ZnS core-multishell quantum dots in human umbilical vein endothelial cells.

Colloidal semiconductor quantum dots (QDs) have been extensively researched and developed for biomedical applications, including drug delivery and biosensing assays. Hence, it is pivotal to understand their behavior in terms of intracellular transport and toxicological effects. In this study, we focused on 3-mercaptopropionic acid-coated CdSe-CdS/ZnS core-multishell quantum dots (3MPA-QDs) converted from the as-grown octadecylamine-coated quantum dots (ODA-QDs) and their direct and dynamic interactions with human umbilical vein endothelial cells (HUVECs). Live cell imaging using confocal fluorescence microscopy showed that 3MPA-QDs first attached to and subsequently aggregated on HUVEC plasma membrane ~25 min after QD deposition. The aggregated QDs started being internalized at ~2 h and reached their highest internalization degree at ~24 h. They were released from HUVECs after ~48 h. During the 48 h period, the HUVECs responded normally to external stimulations, grew, proliferated and wound healed without any perceptible apoptosis. Furthermore, 1) 3MPA-QDs were internalized in newly formed LysoTracker-stained early endosomes; 2) adenosine 5'-triphosphate-induced [Ca2+]i modulation caused a transient decrease in the fluorescence of 3MPA-QDs that were attached to the plasma membrane but a transient increase in the internalized 3MPA-QDs; and 3) fluorescence signal modulations of co-stained LysoTracker and QDs induced by the lysosomotropic agent Gly-Phe-ß-naphthylamide were spatially co-localized and temporally synchronized. Our findings suggest that 3MPA-QDs converted from ODA-QDs are a potential nontoxic fluorescent probe for future use in clinical applications. Moreover, the photophysical strategy and techniques reported in this work are easily applicable to study of direct interactions between other nanoparticles and live cells; contributing to awareness and implementation of the safe applications of nanoparticles.