Cell Biology


Protocols in Current Issue
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0 Q&A 369 Views Jan 5, 2023

Skeletal muscle, one of the most abundant tissue in the body, is a highly regenerative tissue. Indeed, compared to other tissues that are not able to regenerate after injury, skeletal muscle can fully regenerate upon mechanically, chemically, and infection-induced trauma. Several injury models have been developed to thoroughly investigate the physiological mechanisms regulating skeletal muscle regeneration. This protocol describes how to induce muscle regeneration by taking advantage of a cardiotoxin (CTX)-induced muscle injury model. The overall steps include CTX injection of tibialis anterior (TA) muscles of BL6N mice, collection of regenerating muscles at different time points after CTX injury, and histological characterization of regenerating muscles. Our protocol, compared with others such as those for freeze-induced injury models, avoids laceration or infections of the muscles since it involves neither surgery nor suture. In addition, our protocol is highly reproducible, since it causes homogenous myonecrosis of the whole muscle, and further reduces animal pain and stress.

Graphical abstract

0 Q&A 624 Views Nov 5, 2022

Bronchopulmonary dysplasia (BPD) and pulmonary hypertension associated with BPD (BPD-PH) are of multifactorial origin and share common risk factors. Most murine models of BPD expose newborn pups to only one of these risk factors—more commonly postnatal hyperoxia—thereby mimicking the vital increased fraction of inspired oxygen (FiO2) that preterm infants in neonatal intensive care units often require. To improve representation of the multifactorial origins of BPD and BPD-PH, we established a double hit model, combining antenatal systemic inflammation followed by postnatal hyperoxia. On embryonic day 14, pups are exposed to systemic maternal inflammation via a single intraperitoneal injection of 150 µg/kg of lipopolysaccharide to the dam. Within 24 h after birth, pups and dams are randomized and exposed to gas with either an FiO2 of 0.21 (room air) or 0.65 (hyperoxia 65%). In our BPD and BPD-PH double hit model, we can obtain multiple readouts from individual pups that include echocardiography, lung histology and immunohistochemistry, ex vivo X-ray micro computed tomography, and pulmonary and plasmatic immunity by RNA, protein, or flow cytometry.

Graphical abstract:

Figure 1. Murine double hit model of cardiopulmonary disease.
On embryonic day (E)14, pups are exposed to systemic maternal inflammation via a single intraperitoneal injection of 150 µg/kg lipopolysaccharide to the dam. Within 24 h after birth, pups and dams are randomized to be exposed to gas with either a fraction of inspired oxygen (FiO2) of 0.21 (air; 21% O2) or 0.65 (hyperoxia; 65% O2) for a maximum of 28 days. According to the murine stage of lung development (Schittny, 2017), experimental endpoints include postnatal day (D)3, D5, D14, D28, and D60.

0 Q&A 564 Views Oct 5, 2022

Late-gestation transient intrauterine hypoxia is a common cause of birth injury. It can lead to long-term neurodevelopmental disabilities even in the absence of gross anatomic injury. Currently, postnatal models of hypoxia–ischemia are most commonly used to study the effect of oxygen deprivation in the fetal brain. These models, however, are unable to take into account placental factors that influence the response to hypoxia, exhibit levels of cell death not seen in many human patients, and are unable to model preterm hypoxia. To address this gap in research, we have developed a protocol to induce transient hypoxia in fetal mice. A pregnant dam at gestational day 17.5 is placed into a hypoxia chamber. Over 30 min, the inspired oxygen is titrated from 21% (ambient air) to 5%. The dam remains in the chamber for up to 8 h, after which fetal brains can be collected or pups delivered for postnatal studies. This protocol recapitulates phenotypes seen in human patients exposed to transient in utero hypoxia and is readily reproducible by researchers.

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0 Q&A 1350 Views Jul 5, 2022

Work in cold environments may have a significant impact on occupational health. In these and similar situations, cold exposure localized to the extremities may reduce the temperature of underlying tissues. To investigate the molecular effects of cold exposure in muscle, and since adequate methods were missing, we established two experimental cold exposure models: 1) In vitro exposure to cold (18°C) or control temperature (37°C) of cultured human skeletal muscle cells (myotubes); and 2) unilateral cold exposure of hind limb skeletal muscle in anesthetized rats (intramuscular temperature 18°C), with contralateral control (37°C). This methodology enables studies of muscle responses to local cold exposures at the level of gene expression, but also other molecular outcomes.

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0 Q&A 1350 Views Jun 5, 2022

Damage to the plasma membrane and loss of membrane integrity are detrimental to eukaryotic cells. It is, therefore, essential that cells possess an efficient membrane repair system to survive. However, the different cellular and molecular mechanisms behind plasma membrane repair have not been fully elucidated. Here, we present three complementary methods for plasma membrane wounding, and measurement of membrane repair and integrity. The first protocol is based on real time imaging of cell membrane repair kinetics in response to laser-induced injury. The second and third protocols are end point assays that provide a population-based measure of membrane integrity, after either mechanical injury by vortex mixing with glass beads, or by detergent-induced injury by digitonin in sublytic concentrations. The protocols can be applied to most adherent eukaryotic cells in culture, as well as cells in suspension.

0 Q&A 1491 Views Jun 5, 2022

Our ability to move and breathe requires an efficient communication between nerve and muscle that mainly takes place at the neuromuscular junctions (NMJs), a highly specialized synapse that links the axon of a motor neuron to a muscle fiber. When NMJs or axons are disrupted, the control of muscle fiber contraction is lost and muscle are paralyzed. Understanding the adaptation of the neuromuscular system to permanent or transient denervation is a challenge to understand the pathophysiology of many neuromuscular diseases. There is still a lack of in vitro models that fully recapitulate the in vivo situation, and in vivo denervation, carried out by transiently or permanently severing the nerve afferent to a muscle, remains a method of choice to evaluate reinnervation and/or the consequences of the loss of innervation. We describe here a simple surgical intervention performed at the hip zone to expose the sciatic nerve in order to obtain either permanent denervation (nerve-cut) or transient and reversible denervation (nerve-crush). These two methods provide a convenient in vivo model to study adaptation to denervation.

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0 Q&A 1856 Views Dec 20, 2021

Wound healing is a critical process for maintaining the integrity of tissues, driven in large part by the active migration of cells to cover damaged regions. While the long-term tissue injury response over hours and days has been extensively studied, the rapid early migratory response of cells to injury in vivo is still being uncovered, especially in model systems such as zebrafish larvae, which are ideal for live imaging with high spatiotemporal resolution. Observing these dynamics requires a wounding method that prompts a robust wound response and is compatible with immediate live imaging or other downstream applications. We have developed a procedure for wounding the epidermis in the tailfin of larval zebrafish, which we term “tissue laceration”. In this procedure, the tailfin is impaled with a glass needle that is then dragged through the tissue, which generates a full-thickness wound that elicits a dramatic migratory wound response within seconds from cells up to several hundred micrometers away from the wound. Laceration generates a larger wound response in the first few minutes following wounding compared to other mechanical wounds such as tail transection, and laceration does not require specialized equipment compared to laser wounding methods. This procedure can be used to interrogate the processes by which epidermal cells far away from the wound are able to rapidly detect injury and respond to the wound.

0 Q&A 2548 Views Jun 5, 2021

Cryoinjury, or injury due to freezing, is a method of creating reproducible, local injuries in skeletal muscle. This method allows studying the regenerative response following muscle injuries in vivo, thus enabling the evaluation of local and systemic factors that influence the processes of myofiber regeneration. Cryoinjuries are applicable to the study of various modalities of muscle injury, particularly non-traumatic and traumatic injuries, without a loss of substantial volume of muscle mass. Cryoinjury requires only simple instruments and has the advantage over other methods that the extent of the lesion can be easily adjusted and standardized according to the duration of contact with the freezing instrument. The regenerative response can be evaluated histologically by the average maturity of regenerating myofibers as indicated by the cross-sectional areas of myofibers with centrally located nuclei. Accordingly, cryoinjury is regarded as one of the most reliable and easily accessible methods for simulating muscle injuries in studies of muscle regeneration.

0 Q&A 2388 Views Jan 20, 2021

Research on wound healing majorly relies on rat, mice and other animal models. However, an alternative animal model ought to be brought in the field, pertaining to the stringent ethical issues owing to the use of animals in research. In this regard, Caenorhabdits elegans, a miniature model nematode gains the great attention of the researchers in wound healing. Though, the model is being explored in wound research for more than a decade, the existing protocols lack the acquisition of large wound population that in turn could enable the utility of global genomics (G), proteomics (P) and metabolomics (M) based approaches. In order to overcome the inadequacy of the existing protocols, the protocol described here affords the acquisition of voluminous wound population in C. elegans using truncated glasswool pieces to enable the utility of high throughput analytical techniques.

Graphic abstract

Steps involved in glass wool wounding protocol.

0 Q&A 4251 Views Aug 5, 2019
Splenectomy in an animal model requires a standardized technique utilizing best practice to avoid variability which can result in adverse impact to the animal resulting in flawed physiologic responses simply due to technique rather than to the studied variables. In the case of the spleen, often investigators are analyzing the animal immune or inflammatory responses. Surgical splenectomy involves many variables from the training and expertise of the surgeon, which directly correlates to surgical technique to the length of operation and ease of the procedure. This operation, in turn, impacts blood loss and insensible fluid losses, sterile technique, unintended trauma to the spleen and surrounding organs, the length of the incision and the duration of the operation with more prolonged exposure to anesthetic agents. All these variables ultimately play a significant role in the experiment since they directly affect the response of the model in terms of inflammation, immune activation, or even suppression. Undesired variables such as these go unnoticed and lead to inaccurate and misleading data.

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