Cell Biology

In mammals, the skin comprises several distinct cell populations that are organized into the following layers: epidermis (stratum corneum, stratum granulosum, stratum spinosum, and basal layer), basement membrane, dermis, and hypodermal (subcutaneous fat) layers. It is vital to identify the exact location and function of proteins in different skin layers. Laser capture microdissection (LCM) is an effective technique for obtaining pure cell populations from complex tissue sections for disease-specific genomic and proteomic analysis. In this study, we used LCM to isolate different skin layers, constructed a stratified developmental lineage proteome map of human skin that incorporates spatial protein distribution, and obtained new insights into the role of extracellular matrix (ECM) on stem cell regulation.

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

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.

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.

Graphical abstract:

The ex vivo experimentation with surgically discarded human skin represents a unique methodology amenable for mechanism and pharmacologic agent studies without the involvement of human subjects. Here, we describe a protocol that includes preparation, culture, and stimulation of human skin explants, and subsequent analyses by quantitative reverse transcription PCR and immunostaining. This protocol may also be applied for ex vivo studies of murine skin, reducing animal numbers and potentially harmful treatments. In our hands, this protocol has been used for wound healing, viral infection, and hair growth–related studies.

Graphical abstract:

Cartoon of explant skin culture.

Skin explant sits on top of a gelatin surgical sponge saturated with culture medium at an air–liquid interface.

Acute respiratory distress syndrome (ARDS) is a life-threatening, high mortality pulmonary condition characterized by acute lung injury (ALI) resulting in diffuse alveolar damage. Despite progress regarding the understanding of ARDS pathophysiology, there are presently no effective pharmacotherapies. Due to the complexity and multiorgan involvement typically associated with ARDS, animal models remain the most commonly used research tool for investigating potential new therapies. Experimental models of ALI/ARDS use different methods of injury to acutely induce lung damage in both small and large animals. These models have historically played an important role in the development of new clinical interventions, such as fluid therapy and the use of supportive mechanical ventilation (MV). However, failures in recent clinical trials have highlighted the potential inadequacy of small animal models due to major anatomical and physiological differences, as well as technical challenges associated with the use of clinical co-interventions [e.g., MV and extracorporeal membrane oxygenation (ECMO)]. Thus, there is a need for larger animal models of ALI/ARDS, to allow the incorporation of clinically relevant measurements and co-interventions, hopefully leading to improved rates of clinical translation. However, one of the main challenges in using large animal models of preclinical research is that fewer species-specific experimental tools and metrics are available for evaluating the extent of lung injury, as compared to rodent models. One of the most relevant indicators of ALI in all animal models is evidence of histological tissue damage, and while histological scoring systems exist for small animal models, these cannot frequently be readily applied to large animal models. Histological injury in these models differs due to the type and severity of the injury being modeled. Additionally, the incorporation of other clinical support devices such as MV and ECMO in large animal models can lead to further lung damage and appearance of features absent in the small animal models. Therefore, semi-quantitative histological scoring systems designed to evaluate tissue-level injury in large animal models of ALI/ARDS are needed. Here we describe a semi-quantitative scoring system to evaluate histological injury using a previously established porcine model of ALI via intratracheal and intravascular lipopolysaccharide (LPS) administration. Additionally, and owing to the higher number of samples generated from large animal models, we worked to implement a more sustainable and greener histopathological workflow throughout the entire process.

The retina is a thin neuronal multilayer responsible for the detection of visual information. The first step in visual transduction occurs in the photoreceptor outer segment. The studies on photoreception and visual biochemistry have often utilized rod outer segments (OS) or OS disks purified from mammalian eyes. Literature reports several OS and disk purification procedures that rarely specify the procedure utilized to collect the retina from the eye. Some reports suggest the use of scissors, while others do not mention the issue as they declare to utilize frozen retinas. Because the OS are deeply embedded in the retinal pigmented epithelium (RPE), the detachment of the retina by a harsh pull-out can cause the fracture of the photoreceptor cilium. Here, we present a protocol maximizing OS yield. Eye semi-cups, obtained by hemisecting the eyeball and discarding the anterior chamber structures and the vitreous, are filled with Mammalian Ringer. After 10–15 min of incubation, the retinas spontaneously detach with their wealth of OS almost intact. The impressive ability of the present protocol to minimize the number of OS stuck inside the RPE, and therefore lost, compared with the classic procedure, is shown by confocal laser scanning microscopy analysis of samples stained ex vivo with a dye (MitoTracker deep red) that stains both retinal mitochondria and OS. Total protein assay of OS disks purified by either procedure also shows a 300% total protein yield improvement. The advantage of the protocol presented is its higher yield of photoreceptor OS for subsequent purification procedures, while maintaining the physiological features of the retina.

Senescence-associated beta-galactosidase (SA-β-GAL) is an enzyme that accumulates in the lysosomes of senescent cells, where it hydrolyses β-galactosides. With p16, it represents a well-recognized biomarker used to assess senescence both in vivo and in cell culture. The use of a chromogenic substrate, such as 5-bromo-4-chloro-3-indoyl-β-d-galactopyranoside (X-Gal), allows the detection of SA-β-GAL activity at pH 6.0 by the release of a visible blue product. Senescence occurs during aging and is part of the aging process itself. We have shown that prematurely aged zebrafish accumulate senescent cells detectable by SA-β-GAL staining in different tissues, including testis and gut. Here, we report a detailed protocol to perform an SA-β-GAL assay to detect senescent cell accumulation across the entire adult zebrafish organism (Danio rerio). We also identify previously unreported organs that show increased cell senescence in telomerase mutants, including the liver and the spinal cord.

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.

Graphical abstract:

Aging and wasting of skeletal muscle reduce organismal fitness. Regrettably, only limited interventions are currently available to address this unmet medical need. Many methods have been developed to study this condition, including the intramuscular electroporation of DNA plasmids. However, this technique requires surgery and high electrical fields, which cause tissue damage. Here, we report an optimized protocol for the electroporation of small interfering RNAs (siRNAs) into the tibialis anterior muscle of mice. This protocol does not require surgery and, because of the small siRNA size, mild electroporation conditions are utilized. By inducing target mRNA knockdown, this method can be used to interrogate gene function in muscles of mice from different strains, genotypes, and ages. Moreover, a complementary method for siRNA transfection into differentiated myotubes can be used for testing siRNA efficacy before in vivo use. Altogether, this streamlined protocol is instrumental for basic science and translational studies in muscles of mice and other animal models.