Advances in 3D Bioprinted Cardiac Tissue Using Stem Cell-Derived Cardiomyocytes.

The ultimate goal of cardiac tissue engineering is to generate new muscle to repair or replace the damaged heart. This requires advances in stem cell technologies to differentiate billions of cardiomyocytes, together with advanced biofabrication approaches such as 3D bioprinting to achieve the requisite structure and contractile function. In this concise review, we cover recent progress in 3D bioprinting of cardiac tissue using pluripotent stem cell-derived cardiomyocytes, key design criteria for engineering aligned cardiac tissues, and ongoing challenges in the field that must be addressed to realize this goal.

Evaluation of Senescence and Its Prevention in Doxorubicin-Induced Cardiotoxicity Using Dynamic Engineered Heart Tissues.

Background: Doxorubicin is an essential cancer treatment, but its usefulness is hampered by the occurrence of cardiotoxicity. Nevertheless, the pathophysiology underlying doxorubicin-induced cardiotoxicity and the respective molecular mechanisms are poorly understood. Recent studies have suggested involvement of cellular senescence. Objectives: The aims of this study were to establish whether senescence is present in patients with doxorubicin-induced cardiotoxicity and to investigate if this could be used as a potential treatment target. Methods: Biopsies from the left ventricles of patients with severe doxorubicin-induced cardiotoxicity were compared with control samples. Additionally, senescence-associated mechanisms were characterized in 3-dimensional dynamic engineered heart tissues (dyn-EHTs) and human pluripotent stem cell-derived cardiomyocytes. These were exposed to multiple, clinically relevant doses of doxorubicin to recapitulate patient treatment regimens. To prevent senescence, dyn-EHTs were cotreated with the senomorphic drugs 5-aminoimidazole-4-carboxamide ribonucleotide and resveratrol. Results: Senescence-related markers were significantly up-regulated in the left ventricles of patients with doxorubicin-induced cardiotoxicity. Treatment of dyn-EHTs resulted in up-regulation of similar senescence markers as seen in the patients, accompanied by tissue dilatation, decreased force generation, and increased troponin release. Treatment with senomorphic drugs led to decreased expression of senescence-associated markers, but this was not accompanied by improved function. Conclusions: Senescence was observed in the hearts of patients with severe doxorubicin-induced cardiotoxicity, and this phenotype can be modeled in vitro by exposing dyn-EHTs to repeated clinically relevant doses of doxorubicin. The senomorphic drugs 5-aminoimidazole-4-carboxamide ribonucleotide and resveratrol prevent senescence but do not result in functional improvements. These findings suggest that preventing senescence by using a senomorphic during doxorubicin administration might not prevent cardiotoxicity.

3D-bioprinted human tissue and the path toward clinical translation.

Three-dimensional (3D) bioprinting is a transformative technology for engineering tissues for disease modeling and drug screening and building tissues and organs for repair, regeneration, and replacement. In this Viewpoint, we discuss technological advances in 3D bioprinting, key remaining challenges, and essential milestones toward clinical translation.

Recapitulating human cardio-pulmonary co-development using simultaneous multilineage differentiation of pluripotent stem cells.

The extensive crosstalk between the developing heart and lung is critical to their proper morphogenesis and maturation. However, there remains a lack of models that investigate the critical cardio-pulmonary mutual interaction during human embryogenesis. Here, we reported a novel stepwise strategy for directing the simultaneous induction of both mesoderm-derived cardiac and endoderm-derived lung epithelial lineages within a single differentiation of human-induced pluripotent stem cells (hiPSCs) via temporal specific tuning of WNT and nodal signaling in the absence of exogenous growth factors. Using 3D suspension culture, we established concentric cardio-pulmonary micro-Tissues (µTs), and expedited alveolar maturation in the presence of cardiac accompaniment. Upon withdrawal of WNT agonist, the cardiac and pulmonary components within each dual-lineage µT effectively segregated from each other with concurrent initiation of cardiac contraction. We expect that our multilineage differentiation model will offer an experimentally tractable system for investigating human cardio-pulmonary interaction and tissue boundary formation during embryogenesis.

Organs begin developing during the first few months of pregnancy, while the baby is still an embryo. These early stages of development are known as embryogenesis ­ a tightly organized process, during which the embryo forms different layers of stem cells. These cells can be activated to turn into a particular type of cell, such as a heart or a lung cell. The heart and lungs develop from different layers within the embryo, which must communicate with each other for the organs to form correctly. For example, chemical signals can be released from and travel between layers of the embryo, activating processes inside cells located in the different areas. In mouse models, chemical signals and cells travel between developing heart and lung, which helps both organs to form into the correct structure. But it is unclear how well the observations from mouse models translate to heart and lung development in humans. To find out more, Ng et al. developed a human model of heart and lung co-development during embryogenesis using human pluripotent stem cells. The laboratory-grown stem cells were treated with chemical signals, causing them to form different layers that developed into early forms of heart and lung cells. The cells were then transferred into a specific growing condition, where they arranged into three-dimensional structures termed microtissues. Ng et al. found that lung cells developed faster when grown in microtissues with accompanying developing heart cells compared to microtissues containing only developing lung cells. In addition, Ng et al. revealed that the co-developing heart and lung tissues automatically separate from each other during later stage, without the need for chemical signals. This human cell-based model of early forms of co-developing heart and lung cells may help provide researchers with new strategies to probe the underlying mechanisms of human heart and lung interaction during embryogenesis.

Dynamic loading of human engineered heart tissue enhances contractile function and drives a desmosome-linked disease phenotype.

The role that mechanical forces play in shaping the structure and function of the heart is critical to understanding heart formation and the etiology of disease but is challenging to study in patients. Engineered heart tissues (EHTs) incorporating human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes have the potential to provide insight into these adaptive and maladaptive changes. However, most EHT systems cannot model both preload (stretch during chamber filling) and afterload (pressure the heart must work against to eject blood). Here, we have developed a new dynamic EHT (dyn-EHT) model that enables us to tune preload and have unconstrained contractile shortening of >10%. To do this, three-dimensional (3D) EHTs were integrated with an elastic polydimethylsiloxane strip providing mechanical preload and afterload in addition to enabling contractile force measurements based on strip bending. Our results demonstrated that dynamic loading improves the function of wild-type EHTs on the basis of the magnitude of the applied force, leading to improved alignment, conduction velocity, and contractility. For disease modeling, we used hiPSC-derived cardiomyocytes from a patient with arrhythmogenic cardiomyopathy due to mutations in the desmoplakin gene. We demonstrated that manifestation of this desmosome-linked disease state required dyn-EHT conditioning and that it could not be induced using 2D or standard 3D EHT approaches. Thus, a dynamic loading strategy is necessary to provoke the disease phenotype of diastolic lengthening, reduction of desmosome counts, and reduced contractility, which are related to primary end points of clinical disease, such as chamber thinning and reduced cardiac output.

Gain-of-function mutation in ubiquitin-ligase KLHL24 causes desmin degradation and dilatation in hiPSC-derived engineered heart tissues.

The start codon c.1A>G mutation in KLHL24, encoding ubiquitin-ligase KLHL24, results in the loss of 28 N-terminal amino acids (KLHL24-ΔN28) by skipping the initial start codon. In skin, KLHL24-ΔN28 leads to gain of function, excessively targeting intermediate filament keratin-14 for proteasomal degradation, ultimately causing epidermolysis bullosa simplex (EBS). The majority of these EBS-patients are also diagnosed with dilated cardiomyopathy (DCM), but the pathological mechanism in the heart is unknown. As desmin is the cardiac homologue of keratin-14, we hypothesized that KLHL24-ΔN28 leads to excessive degradation of desmin, resulting in DCM. Dynamically loaded engineered heart tissues (dyn-EHTs) were generated from human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes from two patients and three (non)familial controls. Ten-fold lower desmin protein levels were observed in patient-derived dyn-EHTs, in line with diminished desmin levels detected in patients' explanted heart. This was accompanied by tissue dilatation, impaired mitochondrial function, decreased force values and increased cardiomyocyte stress. HEK293 transfection studies confirmed KLHL24-mediated desmin degradation. KLHL24 RNA interference or direct desmin overexpression recovered desmin protein levels, restoring morphology and function in patient-derived dyn-EHTs. To conclude, presence of KLHL24-ΔN28 in cardiomyocytes leads to excessive degradation of desmin, affecting tissue morphology and function, that can be prevented by restoring desmin protein levels.

Adipose Stem Cells Enhance Nerve Regeneration and Muscle Function in a Peroneal Nerve Ablation Model.

Severe peripheral nerve injuries have devastating consequences on the quality of life in affected patients, and they represent a significant unmet medical need. Destruction of nerve fibers results in denervation of targeted muscles, which, subsequently, undergo progressive atrophy and loss of function. Timely restoration of neural innervation to muscle fibers is crucial to the preservation of muscle homeostasis and function. The goal of this study was to evaluate the impact of addition of adipose stem cells (ASCs) to polycaprolactone (PCL) nerve conduit guides on peripheral nerve repair and functional muscle recovery in the setting of a critical size nerve defect. To this end, peripheral nerve injury was created by surgically ablating 6 mm of the common peroneal nerve in a rat model. A PCL nerve guide, filled with ASCs and/or poloxamer hydrogel, was sutured to the nerve ends. Negative and positive controls included nerve ablation only (no repair), and reversed polarity autograft nerve implant, respectively. Tibialis anterior (TA) muscle function was assessed at 4, 8, and 12 weeks postinjury, and nerve and muscle tissue was retrieved at the 12-week terminal time point. Inclusion of ASCs in the PCL nerve guide elicited statistically significant time-dependent increases in functional recovery (contraction) after denervation; ∼25% higher than observed in acellular (poloxamer-filled) implants and indistinguishable from autograft implants, respectively, at 12 weeks postinjury (p < 0.05, n = 7-8 in each group). Analysis of single muscle fiber cross-sectional area (CSA) revealed that ASC-based treatment of nerve injury provided a better recapitulation of the overall distribution of muscle fiber CSAs observed in the contralateral TA muscle of uninjured limbs. In addition, the presence of ASCs was associated with improved features of re-innervation distal to the defect, with respect to neurofilament and S100 (Schwann cell marker) expression. In conclusion, these initial studies indicate significant benefits of inclusion of ASCs to the rate and magnitude of both peripheral nerve regeneration and functional recovery of muscle contraction, to levels equivalent to autograft implantation. These findings have important implications to improved nerve repair, and they provide input for future work directed to restoration of nerve and muscle function after polytraumatic injury. Impact Statement This works explores the application of adipose stem cells (ASCs) for peripheral nerve regeneration in a rat model. Herein, we demonstrate that the addition of ASCs in poloxamer-filled PCL nerve guide conduits impacts nerve regeneration and recovery of muscle function, to levels equivalent to autograft implantation, which is considered to be the current gold standard treatment. This study builds on the importance of a timely restoration of innervation to muscle fibers for preservation of muscle homeostasis, and it will provide input for future work aiming at restoring nerve and muscle function after polytraumatic injury.

Long-gap peripheral nerve repair through sustained release of a neurotrophic factor in nonhuman primates.

Severe injuries to peripheral nerves are challenging to repair. Standard-of-care treatment for nerve gaps >2 to 3 centimeters is autografting; however, autografting can result in neuroma formation, loss of sensory function at the donor site, and increased operative time. To address the need for a synthetic nerve conduit to treat large nerve gaps, we investigated a biodegradable poly(caprolactone) (PCL) conduit with embedded double-walled polymeric microspheres encapsulating glial cell line-derived neurotrophic factor (GDNF) capable of providing a sustained release of GDNF for >50 days in a 5-centimeter nerve defect in a rhesus macaque model. The GDNF-eluting conduit (PCL/GDNF) was compared to a median nerve autograft and a PCL conduit containing empty microspheres (PCL/Empty). Functional testing demonstrated similar functional recovery between the PCL/GDNF-treated group (75.64 ± 10.28%) and the autograft-treated group (77.49 ± 19.28%); both groups were statistically improved compared to PCL/Empty-treated group (44.95 ± 26.94%). Nerve conduction velocity 1 year after surgery was increased in the PCL/GDNF-treated macaques (31.41 ± 15.34 meters/second) compared to autograft (25.45 ± 3.96 meters/second) and PCL/Empty (12.60 ± 3.89 meters/second) treatment. Histological analyses included assessment of Schwann cell presence, myelination of axons, nerve fiber density, and g-ratio. PCL/GDNF group exhibited a statistically greater average area occupied by individual Schwann cells at the distal nerve (11.60 ± 33.01 µm2) compared to autograft (4.62 ± 3.99 µm2) and PCL/Empty (4.52 ± 5.16 µm2) treatment groups. This study demonstrates the efficacious bridging of a long peripheral nerve gap in a nonhuman primate model using an acellular, biodegradable nerve conduit.

Characteristics and Immunomodulating Functions of Adipose-Derived and Bone Marrow-Derived Mesenchymal Stem Cells Across Defined Human Leukocyte Antigen Barriers.

BACKGROUND: Vascularized composite allotransplantation opens new possibilities in reconstructive transplantation such as hand or face transplants. Lifelong immunosuppression and its side-effects are the main drawbacks of this procedure. Mesenchymal stem cells (MSCs) have clinically useful immunomodulatory effects and may be able to reduce the burden of chronic immunosuppression. Herein, we assess and compare characteristics and immunomodulatory capacities of bone marrow- and adipose tissue-derived MSCs isolated from the same human individual across defined human leukocyte antigen (HLA) barriers. MATERIALS AND METHODS: Samples of omental (o.) adipose tissue, subcutaneous (s.c.) adipose tissue, and bone marrow aspirate from 10 human organ donors were retrieved and MSCs isolated. Cells were characterized by flow cytometry and differentiated in three lineages: adipogenic, osteogenic, and chondrogenic. In mixed lymphocyte reactions, the ability of adipose-derived mesenchymal stem cells (ASCs) and bone marrow-derived mesenchymal stem cells (BMSCs) to suppress the immune response was assessed and compared within individual donors. HLA mismatched or mitogen stimulations were analyzed in co-culture with different MSC concentrations. Supernatants were analyzed for cytokine contents. RESULTS: All cell types, s.c.ASC, o.ASC, and BMSC demonstrated individual differentiation potential and cell surface markers. Immunomodulating effects were dependent on dose and cell passage. Proliferation of responder cells was most effectively suppressed by s.c.ASCs and combination with BMSC resulted in highly efficient immunomodulation. Immunomodulation was not cell contact-dependent and cells demonstrated a specific cytokine secretion. CONCLUSION: When human ASCs and BMSCs are isolated from the same individual, both show effective immunomodulation across defined HLA barriers in vitro. We demonstrate a synergistic effect when cells from the same biologic system were combined. This cell contact-independent function underlines the potential of clinical systemic application of MSCs.

Delivery of adipose-derived stem cells in poloxamer hydrogel improves peripheral nerve regeneration.

INTRODUCTION: Peripheral nerve damage is associated with high long-term morbidity. Because of beneficial secretome, immunomodulatory effects, and ease of clinical translation, transplantation with adipose-derived stem cells (ASC) represents a promising therapeutic modality. METHODS: Effect of ASC delivery in poloxamer hydrogel was assessed in a rat sciatic nerve model of critical-sized (1.5 cm) peripheral nerve injury. Nerve/muscle unit regeneration was assessed via immunostaining explanted nerve, quantitative polymerase chain reaction (qPCR), and histological analysis of reinnervating gastrocnemius muscle. RESULTS: On the basis of viability data, 10% poloxamer hydrogel was selected for in vivo study. Six weeks after transection and repair, the group treated with poloxamer delivered ASCs demonstrated longest axonal regrowth. The qPCR results indicated that the inclusion of ASCs appeared to result in expression of factors that aid in reinnervating muscle tissue. DISCUSSION: Delivery of ASCs in poloxamer addresses multiple facets of the complexity of nerve/muscle unit regeneration, representing a promising avenue for further study. Muscle Nerve 58: 251-260, 2018.

Single Implantable FK506 Disk Prevents Rejection in Vascularized Composite Allotransplantation.

BACKGROUND: In vascularized composite allotransplantation, medication nonadherence leads to increased acute rejections. Improving medication adherence would improve overall allograft survival. Regionally delivered immunosuppression, targeted to sites of allorecognition, may reduce or eliminate the need for daily systemic immunosuppression. METHODS: The authors developed biodegradable FK disks containing FK506-loaded double-walled microspheres and tested their efficacy at preventing rejection in a Brown-Norway-to-Lewis rat hindlimb transplantation model. In some experimental group animals, one FK disk was implanted subcutaneously either in native nontransplanted leg or in a transplanted allograft. Regular blood FK506 levels were measured. The endpoint was 180-day allograft survival or grade 3 rejection. At the endpoint, tissue FK506 levels were measured and mixed lymphocytic reaction was performed. RESULTS: A single FK disk maintained systemic blood FK506 levels between 5 and 15 ng/ml for 146 ± 11.1 days. After that, the levels declined to less than 5 ng/ml through the endpoint. There was significantly increased FK506 concentration in groin lymph nodes draining the implanted FK disk. Compared with other groups, animals with an FK disk in the transplanted allograft had 100 percent allograft survival to more than 180 days despite subtherapeutic levels below 5 ng/ml. In these animals, significant T-cell hyporesponsiveness was seen in groin lymph nodes draining the FK disk compared with robust splenic T-cell proliferation. CONCLUSIONS: Sustained regional immunosuppression (with a single FK506 disk) maintained the allograft by means of a high regional concentration of FK506. Notably, this was achieved at subtherapeutic blood concentrations of FK506, without any further systemic FK506 administration.

Delivery of chondroitinase ABC and glial cell line-derived neurotrophic factor from silk fibroin conduits enhances peripheral nerve regeneration.

Nerve conduits are a proven strategy for guiding axon regrowth following injury. This study compares degradable silk-trehalose films containing chondroitinase ABC (ChABC) and/or glial cell line-derived neurotrophic factor (GDNF) loaded within a silk fibroin-based nerve conduit in a rat sciatic nerve defect model. Four groups of silk conduits were prepared, with the following silk-trehalose films inserted into the conduit: (a) empty; (b) 1 µg GDNF; (3) 2 U ChABC; and (4) 1 µg GDNF/2 U ChABC. Drug release studies demonstrated 20% recovery of GDNF and ChABC at 6 weeks and 24 h, respectively. Six conduits of each type were implanted into 15 mm sciatic nerve defects in Lewis rats; conduits were explanted for histological analysis at 6 weeks. Tissues stained with Schwann cell S-100 antibody demonstrated an increased density of cells in both GDNF- and ChABC-treated groups compared to empty control conduits (p < 0.05). Conduits loaded with GDNF and ChABC also demonstrated higher levels of neuron-specific PGP 9.5 protein when compared to controls (p < 0.05). In this study we demonstrated a method to enhance Schwann cell migration and proliferation and also foster axonal regeneration when repairing peripheral nerve gap defects. Silk fibroin-based nerve conduits possess favourable mechanical and degradative properties and are further enhanced when loaded with ChABC and GDNF. Copyright © 2014 John Wiley & Sons, Ltd.

Administration of adipose-derived stem cells enhances vascularity, induces collagen deposition, and dermal adipogenesis in burn wounds.

Current treatment options for severe burn wounds are often insufficient in reconstructing skin and soft tissue defects. Adipose-derived stem cells (ASCs), a readily available source of multipotent stem cells, represent a promising therapy for the treatment of full-thickness burn wounds. Full-thickness burn wounds were created on the paraspinal region of athymic mice. A one-time, sub-eschar injection of 6.8×10(6) ASCs in PBS or PBS alone was administered at 24-h postoperatively. Time to healing was quantified using Image J analysis. At days 4, 7, 14, and 21, mice were sacrificed and tissues were excised for molecular and histological analysis. ASCs were able to survive in burn wounds as determined by the presence of PKH labeling and human PPARγ expression within the wounds. CD-31 staining demonstrated increased vascularity in ASC-treated wounds at POD 4 (p<0.05). Molecular studies showed enhanced adipogenesis, as well as type III and type I collagen deposition in the ASC treated group (p<0.05). An increase in the mRNA expression ratio of type III to type I collagen was also observed following ASC treatment (p<0.05). By enhancing vascularity, collagen deposition, and adipogenesis, ASCs show promise as an adjunctive therapy for the current treatment of full thickness burn wounds.

Imaging the Stromal Vascular Fraction during Soft-Tissue Reconstruction.

BACKGROUND: Although fat grafting is an increasingly popular practice, suboptimal volume retention remains an obstacle. Graft enrichment with the stromal vascular fraction has gained attention as a method of increasing graft retention. However, few studies have assessed the fate and impact of transplanted stromal vascular fraction on fat grafts. In vivo imaging techniques can be used to help determine the influence stromal vascular fraction has on transplanted fat. METHODS: Stromal vascular fraction was labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide (DiR), a near-infrared dye, and tracked in vivo. Proliferation and differentiation of labeled cells were assessed to confirm that labeling did not adversely affect cellular function. Different doses of labeled stromal vascular fraction were tracked within fat grafts over time using the in vivo imaging system. RESULTS: No significant differences in differentiation and proliferation were observed in labeled versus unlabeled cells (p > 0.05). A pilot study confirmed that stromal vascular fraction fluorescence was localized to fat grafts and different cell doses could be distinguished. A larger-scale in vivo study revealed that stromal vascular fraction fluorescence was statistically significant (p < 0.05) between different cell dose groups and this significance was maintained in higher doses (3 × 10(6) and 2 × 10(6) cells/ml of fat graft) for up to 41 days in vivo. CONCLUSIONS: DiR labeling allowed the authors to differentiate between cell doses and confirm localization. This article supports the use of DiR labeling in conjunction with in vivo imaging as a tool for imaging stromal vascular fraction within fat grafts.

Polymeric biomaterials for nerve regeneration: fabrication and implantation of a biodegradable nerve guide.

Optimizing the quantity, quality, and speed of axon regeneration is important in maximizing functional outcomes following peripheral nerve injury. When severed, injured nerves must be able to regenerate and reconnect to the structures they previously controlled within 12-18 months before sensation and motion are permanently lost. Nerve sprouts from the proximal stump will spontaneously migrate toward the distal stump in the event of a nerve transection. However, surgical intervention remains necessary to repair transection injuries. Regeneration becomes particularly troublesome with large gaps, where autologous nerve grafts or nerve guides are used to repair transected nerves. Nerve conduits function as therapeutic adjuncts, guiding axonal regeneration across gap defects. Despite the availability of several FDA-approved nerve conduits, functional outcomes following their use remain less than optimal. Much work has been focused on developing nerve conduits to improve peripheral nerve repair outcomes. This chapter describes fabrication of a poly(caprolactone) nerve guide and demonstrates its use in a rat sciatic nerve model.

Prevalence of endogenous CD34+ adipose stem cells predicts human fat graft retention in a xenograft model.

BACKGROUND: Fat grafting is a promising technique for soft-tissue augmentation, although graft retention is highly unpredictable and factors that affect graft survival have not been well defined. Because of their capacity for differentiation and growth factor release, adipose-derived stem cells may have a key role in graft healing. The authors' objective was to determine whether biological properties of adipose-derived stem cells present within human fat would correlate with in vivo outcomes of graft volume retention. METHODS: Lipoaspirate from eight human subjects was processed using a standardized centrifugation technique and then injected subcutaneously into the flanks of 6-week-old athymic nude mice. Graft masses and volumes were measured, and histologic evaluation, including CD31+ staining for vessels, was performed 8 weeks after transplantation. Stromal vascular fraction isolated at the time of harvest from each subject was analyzed for surface markers by multiparameter flow cytometry, and also assessed for proliferation, differentiation capacity, and normoxic/hypoxic vascular endothelial growth factor secretion. RESULTS: Wide variation in percentage of CD34+ progenitors within the stromal vascular fraction was noted among subjects and averaged 21.3 ± 15 percent (mean ± SD). Proliferation rates and adipogenic potential among stromal vascular fraction cells demonstrated moderate interpatient variability. In mouse xenograft studies, retention volumes ranged from approximately 36 to 68 percent after 8 weeks, with an overall average of 52 ± 11 percent. A strong correlation (r = 0.78, slope = 0.76, p < 0.05) existed between stromal vascular fraction percentage of CD34+ progenitors and high graft retention. CONCLUSION: Inherent biological differences in adipose tissue exist between patients. In particular, concentration of CD34+ progenitor cells within the stromal vascular fraction may be one of the factors used to predict human fat graft retention.

The effects of repeated handling and corticosterone treatment on behavior in an amphibian (Ocoee salamander: Desmognathus ocoee).

Exposure to unpredictable challenges triggers a stress response that helps an animal cope by ensuring energy availability and increasing expression of anti-predator behaviors. At the same time, stress responses typically suppress activities non-essential to immediate survival, such as growth and reproduction. Glucocorticoid hormones are key mediators of the stress response. We measured the effects of repeated exposure to a handling stressor and repeated elevation of plasma levels of the glucocorticoid hormone, corticosterone (CORT) in a terrestrial salamander, Desmognathus ocoee. Subjects were handled daily or treated every day with a dermal patch containing CORT. Compared to control treatments, chronic handling and treatment with CORT both resulted in decreased body weight. Repeated handling, but not treatment with CORT, reduced feeding in females and activity in both males and females. Treatments had no effect on white blood cell differentials. Despite a nonsignificant trend for courtship to be delayed in handled animals, most salamanders in all treatment groups courted and mated. Courtship and mating may be relatively resistant to the effects of repeated handling and elevated plasma CORT because courtship and mating are energetically inexpensive in this species.

Acute stressors increase plasma corticosterone and decrease locomotor activity in a terrestrial salamander (Desmognathus ochrophaeus).

Vertebrates respond to the onset of an acute stressor with an acute increase in plasma glucocorticoids. The increase in plasma glucocorticoids is believed to be adaptive, helping an animal cope until the stressful episode subsides. Although much is known about the effects of chronic elevation of glucocorticoids, far less is known about the role of acute increases in glucocorticoids in mediating stress responses. To better understand the regulation and function of acute increases in plasma glucocorticoids, we measured stress-induced increases in plasma corticosterone (CORT) and the effects of stressors and exogenous CORT on activity in male Allegheny dusky salamanders (Desmognathus ochrophaeus). Capture and handling of field-caught salamanders resulted in an acute elevation of plasma CORT during the nonmating season but not during the mating season. In laboratory-housed salamanders, a handling stressor that simulated capture resulted in decreased locomotor activity. Noninvasive elevation of plasma CORT via dermal patches did not replicate the handling-induced decrease in activity. Together, this work indicated that 1) the CORT response to the acute stressor of capture and handling was seasonally variable, 2) handling induced a decrease in locomotor activity in the laboratory, and 3) acute increases in plasma CORT did not contribute to stress-induced changes in locomotor activity. Future studies using noninvasive methods to elevate plasma CORT should illuminate the role of acute increases in plasma glucocorticoids in coordinating organismal responses to acute stressors.