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Methods in
Molecular Biology 2193
Hiranmoy Das Editor
Wound
Regeneration
Methods and Protocols
METHODS IN MOLECULAR BIOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, UK
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For over 35 years, biological scientists have come to rely on the research protocols and
methodologies in the critically acclaimed Methods in Molecular Biology series. The series was
the first to introduce the step-by-step protocols approach that has become the standard in all
biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by-
step fashion, opening with an introductory overview, a list of the materials and reagents
needed to complete the experiment, and followed by a detailed procedure that is supported
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Wound Regeneration
Methods and Protocols
Edited by
Hiranmoy Das
Department of Pharmaceutical Sciences, Texas Tech University Health Sciences Center, Amarillo, TX, USA
Editor
Hiranmoy Das
Department of Pharmaceutical Sciences
Texas Tech University Health Sciences Center
Amarillo, TX, USA
ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-0716-0844-9 ISBN 978-1-0716-0845-6 (eBook)
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© Springer Science+Business Media, LLC, part of Springer Nature 2021

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Preface
A wound is defined as a disruption in the continuity of any of the bodily tissues due to
external action, typified by a cut, a bruise, a hematoma, microbial infections, or internal
pathological conditions. Wounds that are not being treated or do not heal through the
normal stages of healing turn out to be chronic. Comorbid pathological conditions also play
a critical role in mediating chronic wounds.
The mechanisms of wound healing are as varied as the tissues in which the process is
carried out. Tissue repair following injury is mediated by a number of cellular and molecular
factors; failure of one or more of these components can lead to impeded healing. Because of
the complex nature of wound healing, a variety of models are needed to study this process in
a laboratory setting. Moreover, many novel therapeutic strategies have emerged within the
field of wound regeneration, which must be evaluated extensively in preclinical models.
This book contains chapters discussing a diverse range of topics related to wound
healing. The generation of wounds is used by one group as a tool to study cellular interac-
tions and growth. Other chapters discuss the mechanisms by which different tissues regen-
erate. Several kinds of ischemic wounds are discussed, as well as several potential mechanisms
by which these challenging wounds may potentially be treated. Several options for repairing
corneal wounds are also proposed. A number of emerging technologies are proposed to help
promote wound healing, including miRNA, nanomaterials, biomaterials, and stem cell
therapies. Several chapters provide detailed protocols for generating wounds in animal
models. As a whole, this book provides methods/protocols/information on a broad range
of topics within the field of wound regeneration.
The discussed methods/protocols/information regarding wound model development
and regeneration studies will be very useful in both the academic and industrial fields of
research. A large range of wounds was considered for inclusion in this book. A variety of
methods for in vitro, in vivo, and ex vivo studies are covered in this method book. A variety
of research laboratories, students, and researchers, both from academic and industrial
settings within the wound regeneration field, will find something of interest in this book.
Amarillo, TX, USA Hiranmoy Das
v
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1 Investigating Epidermal Interactions Through an In Vivo Cutaneous

Wound-Healing Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
John L. Zemkewicz, Racheal G. Akwii, Constantinos M. Mikelis,
and Colleen L. Doçi
2 A Murine Incisional Fetal Wound-Healing Model to Study Scarless
and Fibrotic Skin Repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Traci A. Wilgus
3 Development of Cutaneous Wound in Diabetic Immunocompromised Mice
and Use of Dental Pulp–Derived Stem Cell Product for Healing. . . . . . . . . . . . . . 23
Carl Greene and Hiranmoy Das
4 Musculoskeletal Tissue Engineering Using Fibrous Biomaterials . . . . . . . . . . . . . . 31
George Tan, Yingge Zhou, and Dilshan Sooriyaarachchi
5 Cutaneous Wound Generation in Diabetic NOD/SCID Mice
and the Use of Nanofiber-Expanded Hematopoietic Stem Cell Therapy . . . . . . . 41
Sarah Anderson and Hiranmoy Das
6 Contusion Rodent Model of Traumatic Brain Injury:
Controlled Cortical Impact . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Marie-Line Fournier, Tifenn Clément, Justine Aussudre,
Nikolaus Plesnila, André Obenaus, and Jérôme Badaut
7 Evaluation of MicroRNA Therapeutic Potential Using the Mouse In Vivo
and Human Ex Vivo Wound Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Xi Li and Ning Xu Landén
8 Cellular Migration Assay: An In Vitro Technique to Simulate
the Wound Repair Mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
A K M Nawshad Hossian and George Mattheolabakis
9 In Vivo Ear Sponge Lymphangiogenesis Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Racheal G. Akwii, Md S. Sajib, Fatema T. Zahra,
Hanumantha R. Madala, Kalkunte S. Srivenugopal,
and Constantinos M. Mikelis
10 Identification of Rho GEF and RhoA Activation by Pull-Down Assays . . . . . . . . 97
Md S. Sajib, Fatema T. Zahra, Racheal G. Akwii,
and Constantinos M. Mikelis
11 Wound Matrix Stiffness Imposes on Macrophage Activation . . . . . . . . . . . . . . . . . 111
Pu Duann and Pei-Hui Lin
12 Generation of Acute Hind Limb Ischemia in NOD/SCID Mice
and Treatment with Nanofiber-Expanded CD34+
Hematopoietic Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Derek Barthels and Hiranmoy Das
vii
viii Contents
13 Electrospun Aligned Coaxial Nanofibrous Scaffold for Cardiac Repair . . . . . . . . . 129

Divya Sridharan, Arunkumar Palaniappan,
Britani N. Blackstone, Heather M. Powell,
and Mahmood Khan
14 Generation of Myocardial Ischemic Wounds and Healing
with Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
Daniela Rolph and Hiranmoy Das
15 Corneal Repair Models in Mice: Epithelial/Mechanical
Versus Stromal/Chemical Injuries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
Peipei Pan and Matilda F. Chan
16 Topical Adoptive Transfer of Plasmacytoid Dendritic Cells
for Corneal Wound Healing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
Arsia Jamali, Brendan M. Kenyon, Gustavo Ortiz,
Betul N. Bayraktutar, Victor G. Sendra,
and Pedram Hamrah
17 Murine Corneal Epithelial Wound Modeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
Dhara Shah and Vinay Kumar Aakalu
18 Infection-Induced Porcine Ex Vivo Corneal Wound Model
to Study the Efficacy of Herpes Simplex Virus-1 Entry
and Replication Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
Tejabhiram Yadavalli, Raghuram Koganti,
and Deepak Shukla
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
Contributors
VINAY KUMAR AAKALU • Department of Ophthalmology and Visual Sciences, University of

Illinois at Chicago, Chicago, IL, USA
RACHEAL G. AKWII • Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech
University Health Sciences Center, Amarillo, TX, USA; Department of Pharmaceutical
Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center,
Amarillo, TX, USA
SARAH ANDERSON • Department of Pharmaceutical Sciences, Jerry H. Hodge School of
Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX, USA
JUSTINE AUSSUDRE • CNRS UMR5287, INCIA, University of Bordeaux, Bordeaux, France
JÉRÔME BADAUT • CNRS UMR5287, INCIA, University of Bordeaux, Bordeaux, France;
Department of Basic Sciences, Loma Linda University School of Medicine, Loma Linda,
CA, USA
DEREK BARTHELS • Department of Pharmaceutical Sciences, Jerry H. Hodge School of
BETUL N. BAYRAKTUTAR • Center for Translational Ocular Immunology, Tufts Medical
Center, Tufts University School of Medicine, Boston, MA, USA; Department of
Ophthalmology, Tufts Medical Center, Tufts University School of Medicine, Boston, MA,
USA; Cornea Service, Tufts New England Eye Center, Boston, MA, USA
BRITANI N. BLACKSTONE • Department of Materials Science and Engineering, The Ohio State
University, Columbus, OH, USA
MATILDA F. CHAN • Department of Ophthalmology, School of Medicine, University of
California, San Francisco, CA, USA; Francis I. Proctor Foundation, University of
California, San Francisco, CA, USA
TIFENN CLÉMENT • CNRS UMR5287, INCIA, University of Bordeaux, Bordeaux, France
HIRANMOY DAS • Department of Pharmaceutical Sciences, Jerry H. Hodge School of
COLLEEN L. DOÇI • College of Health Professions, Marian University Indianapolis,
Indianapolis, IN, USA
PU DUANN • Research and Development, Salem Veteran Affairs Medical Center, Salem, VA,
USA
MARIE-LINE FOURNIER • CNRS UMR5287, INCIA, University of Bordeaux, Bordeaux,
France
CARL GREENE • Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy,
Texas Tech University Health Sciences Center, Amarillo, TX, USA
PEDRAM HAMRAH • Center for Translational Ocular Immunology, Tufts Medical Center,
Tufts University School of Medicine, Boston, MA, USA; Department of Ophthalmology,
Tufts Medical Center, Tufts University School of Medicine, Boston, MA, USA; Program in
Neuroscience, Graduate Biomedical Sciences, Tufts University, Boston, MA, USA; Cornea
Service, Tufts New England Eye Center, Boston, MA, USA
A K M NAWSHAD HOSSIAN • Department of Basic Pharmaceutical and Toxicological Sciences,
College of Pharmacy, University of Louisiana Monroe, Monroe, LA, USA
ix
x Contributors
ARSIA JAMALI • Center for Translational Ocular Immunology, Tufts Medical Center, Tufts
University School of Medicine, Boston, MA, USA; Department of Ophthalmology, Tufts
Medical Center, Tufts University School of Medicine, Boston, MA, USA
BRENDAN M. KENYON • Center for Translational Ocular Immunology, Tufts Medical Center,
Tufts Medical Center, Tufts University School of Medicine, Boston, MA, USA; Program in
Neuroscience, Graduate Biomedical Sciences, Tufts University, Boston, MA, USA
MAHMOOD KHAN • Department of Emergency Medicine, The Ohio State University Wexner
Medical Center, Columbus, OH, USA; Dorothy M. Davis Heart & Lung Research
Institute, The Ohio State University Wexner Medical Center, Columbus, OH, USA;
Department of Physiology and Cell Biology, The Ohio State University Wexner Medical
Center, Columbus, OH, USA
RAGHURAM KOGANTI • Department of Ophthalmology and Visual Sciences, University of
NING XU LANDÉN • Dermatology and Venereology Division, Department of Medicine Solna,
Center for Molecular Medicine, Karolinska Institute, Stockholm, Sweden; Ming Wai Lau
Centre for Reparative Medicine, Stockholm Node, Karolinska Institute, Stockholm, Sweden
PEI-HUI LIN • Davis Heart and Lung Research Institute, The Ohio State University,
Columbus, OH, USA; Department of Surgery, The Ohio State University, Columbus, OH,
USA
XI LI • Dermatology and Venereology Division, Department of Medicine Solna, Center for
Molecular Medicine, Karolinska Institute, Stockholm, Sweden
HANUMANTHA R. MADALA • Department of Pharmaceutical Sciences, Jerry H. Hodge School
of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX, USA
GEORGE MATTHEOLABAKIS • Department of Basic Pharmaceutical and Toxicological Sciences,
College of Pharmacy, University of Louisiana Monroe, Monroe, LA, USA
CONSTANTINOS M. MIKELIS • Department of Pharmaceutical Sciences, School of Pharmacy,
Texas Tech University Health Sciences Center, Amarillo, TX, USA; Department of
Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health
Sciences Center, Amarillo, TX, USA
ANDRÉ OBENAUS • Department of Pediatrics, University of California, Irvine, Irvine, CA,
USA; Department of Basic Sciences, Loma Linda University School of Medicine, Loma
Linda, CA, USA
GUSTAVO ORTIZ • Center for Translational Ocular Immunology, Tufts Medical Center, Tufts
University School of Medicine, Boston, MA, USA; Department of Ophthalmology, Tufts
Medical Center, Tufts University School of Medicine, Boston, MA, USA; Cornea Service,
Tufts New England Eye Center, Boston, MA, USA
ARUNKUMAR PALANIAPPAN • Department of Emergency Medicine, The Ohio State University
Wexner Medical Center, Columbus, OH, USA; Centre for Biomaterials, Cell and
Molecular Theranostics, Vellore Institute of Technology, Vellore, India
PEIPEI PAN • Department of Ophthalmology, School of Medicine, University of California,
San Francisco, CA, USA
NIKOLAUS PLESNILA • Institute for Stroke and Dementia Research (ISD), University of
Munich Medical Center, Munich, Germany
HEATHER M. POWELL • Department of Materials Science and Engineering, The Ohio State
University, Columbus, OH, USA; Department of Biomedical Engineering, The Ohio State
University, Columbus, OH, USA; Research Department, Shriners Hospitals for Children,
Cincinnati, OH, USA
Contributors xi
DANIELA ROLPH • Department of Pharmaceutical Sciences, Jerry H. Hodge School of

MD S. SAJIB • Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy,
Texas Tech University Health Sciences Center, Amarillo, TX, USA
VICTOR G. SENDRA • Center for Translational Ocular Immunology, Tufts Medical Center,
Tufts Medical Center, Tufts University School of Medicine, Boston, MA, USA
DHARA SHAH • Department of Ophthalmology and Visual Sciences, University of Illinois at
Chicago, Chicago, IL, USA
DEEPAK SHUKLA • Department of Ophthalmology and Visual Sciences, University of Illinois at
Chicago, Chicago, IL, USA; Department of Microbiology and Immunology, University of
DILSHAN SOORIYAARACHCHI • Department of Industrial, Manufacturing, and Systems
Engineering, Texas Tech University, Lubbock, TX, USA
DIVYA SRIDHARAN • Department of Emergency Medicine, The Ohio State University Wexner
Medical Center, Columbus, OH, USA; Dorothy M. Davis Heart & Lung Research
Institute, The Ohio State University Wexner Medical Center, Columbus, OH, USA
KALKUNTE S. SRIVENUGOPAL • Department of Pharmaceutical Sciences, Jerry H. Hodge School
of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX, USA
GEORGE TAN • Department of Industrial, Manufacturing, and Systems Engineering, Texas
Tech University, Lubbock, TX, USA
TRACI A. WILGUS • Department of Pathology, The Ohio State University, Columbus, OH,
USA
TEJABHIRAM YADAVALLI • Department of Ophthalmology and Visual Sciences, University of
FATEMA T. ZAHRA • Department of Pharmaceutical Sciences, Jerry H. Hodge School of
JOHN L. ZEMKEWICZ • College of Osteopathic Medicine, Marian University Indianapolis,
Indianapolis, IN, USA
YINGGE ZHOU • Department of Industrial, Manufacturing, and Systems Engineering, Texas
Tech University, Lubbock, TX, USA
Chapter 1
Investigating Epidermal Interactions Through an In Vivo

Cutaneous Wound-Healing Assay
John L. Zemkewicz, Racheal G. Akwii, Constantinos M. Mikelis,
and Colleen L. Doçi
Abstract
Cutaneous wound healing is an intricate and multifaceted process. Despite these complexities, the distinct
phases of wound healing provide a unique opportunity to evaluate the roles of different targets in these
coordinated responses. This protocol details an in vivo wound healing assay to study the intersection of
cellular, molecular, and systemic effector pathways. The role of certain proteins in the wound healing
process can be efficiently explored in vivo through the generation of tissue-specific deficient mice. This
approach, although optimized for use with animal models displaying epithelial deficiencies, can be used for
other tissue-specific deficiencies, and utilizes simple and cost-effective methods, allowing investigators to
precisely devise their experimental design. The coordination of immunological, epithelial, vascular, and
microenvironmental factors in wound healing makes this technique a valuable tool for investigators across
fields.
Key words Wound healing, Microenvironment, Epidermal
1 Introduction
Cutaneous wound healing is an intricate and multifaceted process.

Healing requires the coordination of epithelial and vascular
biology, signaling, stem cells, and immunological responses. Recent
studies highlight the sophisticated interplay within the wound
microenvironment, resulting in an increased demand for targeted
methods capable of dissecting these mechanisms. Here, we present
an in vivo wound-healing assay that studies the intersection of
cellular, molecular, and systemic effector pathways. This protocol
is optimized for use with animal models displaying epithelial defi-
ciencies and investigates the function of certain genes in the process
and the relationship between specific molecules and the wound
microenvironment, making this assay a valuable tool for investiga-
tors across fields.
Hiranmoy Das (ed.), Wound Regeneration: Methods and Protocols, Methods in Molecular Biology, vol. 2193,
https://doi.org/10.1007/978-1-0716-0845-6_1, © Springer Science+Business Media, LLC, part of Springer Nature 2021
1
2 John L. Zemkewicz et al.
One of the most robust elements of this assay resides in the

ability to discern varied cellular and molecular roles of targets.
These interactions are frequently absent or superficial in in vitro
assays or profoundly complex and difficult to be conclusively
defined in other in vivo studies. An advantage of this approach to
in vivo wound healing is that it allows the investigator to evaluate
their target of interest in the context of four distinct physiologic
phases, each with their own discrete cellular and molecular events.
These phases include hemostasis, inflammation, proliferation, and
remodeling. While these phases can overlap and may vary in differ-
ent genetic models, the distinct physiological processes housed
within each phase can elucidate multifaceted functions of targets
and their interactions [1].
Wound healing begins immediately after the skin is broken with
coagulation and hemostasis and relies on the interaction between
platelets, collagen, and other components of the extracellular
matrix. In addition to driving clotting, the platelets also release
cytokines and growth factors that stimulate vasodilation, vascular
permeability, and leukocyte recruitment [2]. The second phase of
the wound-healing process is the inflammatory phase, character-
ized by early and late stages. The early inflammatory phase initiates
the innate immune response and recruits neutrophils, which in turn
recruit additional inflammatory cells to the wound microenviron-
ment [3]. Recruitment of macrophages signals the beginning of the
late inflammatory phase, typically anywhere between one and
three days of post-wounding. Macrophages contribute to clearing
wound debris, chemokine secretion, cellular recruitment, and initi-
ation of vascular remodeling [4]. The third phase of the healing
process is the proliferative phase, which begins about 72 h after the
skin is wounded and lasts between one and two weeks. The pro-
liferative phase encompasses multiple physiological processes,
including epithelialization, angiogenesis, signaling, and the prolif-
eration and migration of epithelial cells to fill the wound [5]. Exten-
sive remodeling of the extracellular matrix and neovascularization
drive the formation of granular tissue and promote keratinocyte
migration and proliferation to close the wound. The fourth phase
of the wound-healing process is the remodeling phase, which
involves the development of new epithelium and scar tissue forma-
tion [6]. This phase is typically initiated during the third week of
wound healing but can progress for years. During remodeling,
wounds increase the collagenous extracellular matrix, decrease in
vascular flow, limit metabolic processes, and slow cellular
growth [2].
While these phases house distinct cellular processes, successful
closure and healing of wound rely on effective, coordinated
mechanisms. Disruptions of critical cellular mediators in wound
healing lead to readily observable defects not only in the rate of
wound closure but also in the physiologic presentation of healing
Epidermal Interactions in Wound Healing 3
[7]. Non-healing wounds, chronic ulcers, hypertrophic scars,

keloids, and even tumor development have been observed upon
disruption of the finely tuned balance that contributes to appropri-
ate healing mechanisms [8–10]. These defects have also been
observed in specific cell types. For example, targeted deletion of
Gαq from epidermal cells results in defects in keratinocyte migra-
tion and differentiation and subsequently delays wound healing
[11]. Similarly, loss of Rac1, CXCR2, and other inflammatory and
migratory signaling factors have delayed wound healing through
unique mechanisms [12–14]. Accelerated wound healing has also
been observed in some transgenic models, including those with
deletion of PAI-1 and tropomyosin [15, 16].
Protocols that utilize cutaneous wound healing as a tool for
investigation vary greatly in the literature and can include punch
biopsies, cutaneous flaps, and windows for grafting. However,
many of these methods require extensive technical skills, equip-
ment, or function best in the context of treatment interventions
such as cell transplants, tissue engineering, and topical drug deliv-
ery [17–19]. The protocol presented here represents a simple and
standardized approach that does not require specialized equipment
and can produce robust statistical data for wound-healing altera-
tions throughout the different phases of the process. As such, this
particular iteration of the method is optimal for use in animal
models with epithelial deficiencies.
2 Materials
Animal experiments should take place according to the Institu-

tional Animal Care and Use Committee (IACUC)-approved pro-
tocols of the host institution, in compliance with the Guide for the
Care and Use of Laboratory Animals. The mouse models used in
any experiment may have significant impacts and can vary signifi-
cantly, so investigators should carefully review their models and
implement appropriate controls before starting the wound-healing
protocol. This may require additional materials to those materials
listed below.
2.1 Preparation of 1. Isoflurane or other inhalation anesthetic.

Animals for Wounding 2. Animal hair clippers, preferably no more than 30 mm in width.
2.2 Wound Incision 1. Isoflurane inhalation anesthetic with the appropriate anesthesia
induction chamber, typically regulated for 0.5–2% isoflurane
and 1 L/min flow.
2. Permanent marker.
3. Ruler.
4. Iodopovidone or other topical antiseptic.
5. Scalpel #11 blade, one per three animals to be wounded (see

Note 1).
6. Calipers.
7. Camera.
2.3 Wound 1. Isoflurane or other inhalation anesthetic.

Measurement 2. Calipers.
3. Camera.
2.4 Analysis of 1. Graphing or statistical software package (see Note 2).

Wound Closure
2.5 Preparation of 1. Materials for appropriate euthanasia.

Wounds for 2. Filter paper, approximately 50 mm by 70 mm strips, any
Histological Analysis weight.
3. Sharp scissors or razor blades.
3 Methods
3.1 Preparation of 1. Separate animals for use in this study. Wounded animals can be
Animals for Wounding cohoused, but should not be kept with non-wounded mice (see
Note 3).
2. For inducible animal models, add induction agent (i.e., tamox-
ifen) minimally 1 week prior to wounding (see Note 4).
3. Briefly anesthetize the animals using inhaled isoflurane.
4. Using small clippers, shave a clean rectangle of skin approxi-
mately 30 mm 50 mm (see Note 5).
5. Return the animals to their cage and allow them to recover for
24 h.
3.2 Wound Incision 1. Approximately 24 h after shaving, briefly anesthetize animals

(See Note 6) using inhaled isoflurane.
2. Carefully inspect the animals for any nicks, scratching, or other
evidence of trauma and remove any animals with these signs
from the experimental cohort (Fig. 1, see Note 7).
3. Using a ruler and marker, carefully draw two lines 15 mm apart
on the shaved skin (Fig. 2a).
4. Liberally apply iodopovidone to the skin using a cotton swab.
5. Taking the index finger and thumb of both hands, the first
investigator should pinch the skin just outside the drawn marks
and roll gently back and forth (Fig. 2b, see Note 8). This rolling
motion is repeated until only a thin band of the epidermis is
held taut between the fingers and thumb (Fig. 2c).
Fig. 1 Inspecting animals for injury post-shaving. Animals showing signs of

trauma after shaving can include nicks (a, red arrowheads) and scratching or
skin irritation (b, red arrowheads)
Fig. 2 Procedure for creating individual, replicable wounds. (a) Mice are shaved,
and a 15-mm long incision line is marked. (b) The dorsal skin is pinched
between the forefingers. (c) The skin is rolled repeatedly, until a full-thickness
cutaneous wound without involvement of the underlying muscle layers is possi-
ble. (d) Appearance of a fresh wound using this approach
Fig. 3 Variations in wound appearance. Animals included in the study should have relatively even wounds with
no additional trauma. (a) A clean, well-incised wound. (b) A double wound caused by improper rolling of the
epidermis or incising with the scalpel at an angle. (c) A puncture wound, usually caused by slicing too deeply
upon the initial incision
6. Holding a scalpel vertically, the second investigator should

make a small incision that extends from one of the drawn
lines to the other, exactly 15 mm (Fig. 2d, see Notes 1 and 9).
7. Release the skin and inspect the animal for any punctures, tears,
or cuts outside of the principle wound (Fig. 3, see Note 10).
8. Using calipers, measure the wound length and diameter at its
widest points (Fig. 4 and see Note 11).
9. Photograph each animal with a ruler for reference in each
image.
10. Return the animal to its cage for recovery (see Note 12).
3.3 Wound 1. Carefully anesthetize the animals (see Note 14).

Measurement (See 2. Using calipers, measure the wound length and diameter at its
Note 13) widest points (Fig. 4 and see Note 11). It is recommended that
the same investigator measures each time to ensure that sub-
jective evaluations are consistent (see Note 6).
3. Photograph each animal with a ruler for reference in each
image.
4. Return animals for recovery.
3.4 Analysis of 1. For each of the animals, calculate the total wound area as the
Wound Closure length multiplied by the width of the wound, measured at its
widest dimensions.
2. Normalize to either the measurement taken at the time of
wounding or the measurement taken 12–24 h post-incision
(see Note 15).
Fig. 4 Measurement of incisional wounds. Wounds can take on asymmetrical

boundaries during the healing process. Establishing a consistent protocol for
how wounds are measured is critical for reproducibility, and measuring at the
widest dimensions is recommended. The same wound is shown in (a, b) and in
(c, d). The recommended length and width measurements are indicated by the
dashed line
3. Record when the wound is closed (see Note 16).

4. Investigators should consider including an analysis of wound
half-life, determined from the nonlinear regression of wound
closure over time (see Note 2).
3.5 Preparation of 1. Remove the mouse from the study at the desired time point for
Wounds for analysis (see Note 14).
Histological Analysis 2. Euthanize the mouse according to standard procedures and
protocols.
3. Using sharp scissors or a razor blade, cut a square of skin with
approximately 10–15 mm of normal skin adjacent to the
wound (Figs. 5a, b and see Note 17).
4. Carefully place the skin with the subcutaneous layer facing
down onto a piece of filter paper.
Fig. 5 Harvesting wound samples for histologic analysis. Samples from (a) recently wounded or (b) healed
animals can be collected by first harvesting normal adjacent skin from wound, cutting a 30–40 mm rectangle
around the wound (dashed box). (c) The skin should be mounted on filter paper (gray) and then cut into sagittal
slices for histological processing (dashed lines)
5. Using sharp scissors or a razor blade, carefully cut the paper and
skin into sagittal strips through the length of the wound
(Fig. 5c and Note 18).
6. Proceed with appropriate histological processing for the strips.
4 Notes
1. The scalpel type depends largely on the preference of the

investigator. We have found the #11 scalpel, with the elongated
triangular blade, to be more accurate at making the shallow,
specific cuts needed. However, the shape has less impact on the
outcome than the sharpness of the blade. For that reason, we
recommend using a fresh blade after every third animal.
2. For our analyses, we have used Microsoft Excel and GraphPad
Prism. Other options, including SigmaPlot, R, or any other
software capable of determining a nonlinear regression, are
suitable.
3. Many factors can influence the selection of mice for wound-
healing protocols. Some key considerations should include age,
gender, and background as each can impact the mechanism and
thereby the rate of wound healing. We have found that some
cohorts of animals, such as the Gα11/Gαq eKO previously
reported [11], do not show significant differences in wound
healing after approximately five months of age. Conversely, we
have found in other models with genetic alterations, significant
differences emerge when the animals are one year or older.
Investigators have reported differences in rates of wound heal-
ing among male and female animals [20, 21]. Finally, there are
significant differences influenced by genetic background, par-

ticularly in animals whose immunogenic responses drive
wound-healing mechanisms. In these cases, differences
between BALB/c, C57BL6, or outbred strains such as CD1
had major consequences for healing, which should be consid-
ered. It is recommended therefore that any study using geneti-
cally modified animals should include littermate controls of the
same sex.
4. The length of time for induction or treatment may vary
depending on the systems impacted by the transgenic model.
Full expression of the genetic variation should be confirmed in
the relevant tissues before wounding.
5. Some animals have different hair density, length, and growth
rates, and the average size of the shaved area should be large
enough to prevent excessive hair in the wound area. For some
transgenic or outbred lines, such as CD1, this may require
shaving a significantly larger area than for other genotypes
and backgrounds.
6. Subheading 3.2 requires two investigators. While other parts of
the protocol can be done individually, it is recommended that
the entirety of the technique be done in pairs to maximize
efficiency and minimize the time animals spend under anesthe-
sia. This also allows for the blinding of the study to reduce bias.
7. Some animals may be nicked during the shaving process or
scratch deeply at freshly shaven skin. Animals that have these
types of injuries have already initiated wound-healing mechan-
isms and should be excluded from the study.
8. The rolling method helps ensure the separation of the epider-
mal layer of skin from the muscle layers below it and is essential
for ensuring that animals are wounded in an equivalent and
replicable manner. When rolling, keeping the skin relatively
even is important, as having it slightly twisted can lead to
uneven incisions and further injuries to the mouse.
9. It is important to keep the scalpel completely vertical and make
the incision as shallow as possible. This can avoid double
wounding or tearing, as opposed to cutting, of the skin
(Fig. 3).
10. As with shaving-induced injuries (see Note 7), additional
wounds outside of the principle incision alter the wound-heal-
ing mechanisms enacted by these animals, and they should not
be included in the study.
11. While good technique should result in fairly uniform incisions,
this is not always the case (see Notes 8–10). Measuring at the
widest and longest dimensions of the incision ensures consis-
tent analysis of all wounds across the experimental cohort
(Fig. 4).
12. Animals recover from the procedure quickly, and after a brief
period, do not demonstrate signs of distress. Animals that fail
to recover promptly should be evaluated by a veterinarian or
member of the animal care staff.
13. Animals should be measured every 12–24 h for the first
three days, after which animals can be measured every other
day. Immediately after the incision, the epidermal layer will
show some elasticity in closing, and the first recovery measure-
ment will reflect a combination of skin elasticity and wound
healing. The most rapid rate of wound closure will occur
during the inflammatory phase, and animals should be moni-
tored more closely during this time. Once animals have moved
into the matrix remodeling and vascularization phases, the rate
of wound closure decelerates, and measurements can be made
less frequently.
14. The incisions often overlap regions where investigators would
typically lift, immobilize, and handle the animals, so it is essen-
tial to carefully remove the animals from the cage, anesthetize
the animals, and limit handling as much as possible to avoid
further trauma to the incisions.
15. The normalization of the measurements is recommended but
not necessary for comparison among animal cohorts. In our
experience, individual differences between animals are mini-
mized by normalizing the wound area, but the outcomes are
comparable between both methods when good technique is
present. Thus, it is recommended to identify each mouse
included in the study and to maintain records for each individ-
ual mouse.
16. How wound closure is defined may vary between investigators.
Formation of scar tissue may occur in some animal models that
can make it difficult to distinguish between a closed wound and
a fully healed one. We recommend using a threshold measure-
ment as a definition of closure, often a wound area of less than
5% of the initial wound area.
17. We recommend using sharp scissors rather than razor blades to
isolate and prepare the samples to avoid any tearing, but careful
technique with sharp razor blades or scalpels is also
appropriate.
18. Mounting the skin onto filter paper allows the investigator to
evaluate multiple different cross-sections of the wound on the
same slide. The thickness of the filter paper can vary and
depends largely on the downstream processing of the samples.
Lengthwise cuts through the tissue are essential to preserve the
epithelial tongue and other histologic markers of the prolifera-
tion phase.
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Chapter 2
A Murine Incisional Fetal Wound-Healing Model to Study

Scarless and Fibrotic Skin Repair
Traci A. Wilgus
Abstract
The ideal response to skin injury is the complete regeneration of normal tissue without scar formation. This
regenerative response is known to occur at early stages of embryonic development but is lost as the skin
becomes more mature. In more developed skin, the wound-healing response is suboptimal and results in
the formation of scar tissue. Scar tissue can be a significant clinical concern, causing skin dysfunction as well
as psychosocial issues related to poor aesthetic outcomes. Mouse models of fetal wound healing can be
useful for understanding what regulatory pathways lead to skin regeneration and scarless healing in less
developed skin or scarring and fibrotic healing in more developed skin. Here, a reproducible incisional
wound model in developing mice is described that our lab has used repeatedly to study scarless and fibrotic
fetal wound healing.
Key words Scarless, Regeneration, Wound healing, Fetal, Skin, Development
1 Introduction
The repair of wounds in developed skin is an intricate process that

involves many different cell types and regulatory mediators. This
process is often divided into several stages, including hemostasis/
inflammation, proliferation, and scar formation/remodeling [1–
3]. The ultimate result in this situation is the production and
remodeling of collagen and other extracellular matrix components
by dermal fibroblasts to form scar tissue. Scar tissue acts as a quick
fix to repair damage in the dermal layer of the skin, but there are
several drawbacks to the production of scar tissue as opposed to the
generation of normal dermal tissue. The collagen making up scar
tissue is disorganized and is not aligned or assembled in the same
way as normal dermal collagen, making scar tissue structurally
weaker than the original tissue. In addition, scar tissue has func-
tional deficiencies compared to normal skin, and the aesthetic issues
that come with scarring can lead to a decrease in the quality of life
for many patients [4].
Hiranmoy Das (ed.), Wound Regeneration: Methods and Protocols, Methods in Molecular Biology, vol. 2193,
https://doi.org/10.1007/978-1-0716-0845-6_2, © Springer Science+Business Media, LLC, part of Springer Nature 2021
13
14 Traci A. Wilgus
While scar tissue is a concern with healing in mature skin, fetal

skin can heal via a regenerative, scarless healing process at certain
stages of development [5–7] (Fig. 1). At early gestational stages in
mammals, the response to skin injury is characterized by minimal, if
any, inflammation, a rapid proliferative phase with accelerated
wound closure, and complete regeneration of normal skin tissue
with the absence of scarring [8–10]. As fetal skin becomes more
developed, it gradually loses the ability to undergo regenerative,
scarless healing and begins to respond to wounds as mature skin
would—with a prominent inflammatory response and subsequent
scar formation (fibrotic healing). The transition from scarless to
fibrotic healing takes place at around the third trimester in large
animal models and in human skin [6, 11, 12].
Various animal models have been used to learn more about
scarless fetal healing and about what contributes to the transition
from scarless to fibrotic healing seen during development, includ-
ing murine models [13–17]. Mice are a particularly useful model, as
the gestation length is relatively short (18–21 days), there are
abundant reagents and tools available to study mice, and trans-
genic/knockout mouse strains can be utilized to study the role of
specific mediators or cell types in fetal wound healing in vivo. Our
lab has used a murine model of incisional wound healing to study
the response of fetal skin to injury for many years [10, 17–21]. In
mice, studies have shown that the transition occurs at approxi-
mately embryonic day 16 (E16) [14, 17, 22, 23]. Therefore,
wounds created time points before and after this age can be used
to study scarless (E15) or fibrotic (E18) wound healing in fetal skin
(Fig. 1).
The method outlined in this chapter has been used to generate
cutaneous wounds in utero with the intention of studying the
differences between scarless and fibrotic healing. Comparison of
E15 (scarless) and E18 (fibrotic) wounds can be used to help define
the mechanistic differences between these two distinct types of
healing and to identify potential pro- or anti-fibrotic regulatory
pathways. In addition, the pro-fibrotic potential of various media-
tors can be tested by exposing E15 wounds to the mediator in
question and observing whether the wounds, which would nor-
mally heal in a scarless manner, heal with a scar. Ultimately, study-
ing the scarless fetal wound-healing process using in vivo models
could help shed light on novel strategies to enhance healing and
minimize scarring.
2 Materials
2.1 Animals 1. FVB mice (or similar strain).

2. 8–10-week-old mice.
Murine Incisional Fetal Wound Healing Model 15
Fig. 1 Healing outcomes in murine fetal wounds. (a) The wound-healing response in fetal skin changes during
development. In mice, the skin is able to regenerate and heals without a scar at early stages of development
(until about E16). Then, there is a transition period after which skin wounds lose the ability to heal scarlessly
and instead heal with a scar. (b, c) Representative Masson’s trichrome-stained histological sections of 7-day
wounds generated at E15 (b) or E18 (c). Note the regeneration of hair follicles within the healed wound and
lack of scar tissue in E15 wounds, which is indicative of regenerative, scarless healing. Scale bar ¼ 100 μm;
arrows mark margins of the healed wound or scar
2.2 Surgical 1. Forceps (fine-tipped, toothed, curved, and blunt-end forceps

Instruments, may be needed).
Equipment, 2. Scissors.
and Materials
3. Microsurgical scissors.
4. Needle holder.
5. Hamilton syringe (10 μl), with removable needle.
6. Gauze (nonwoven).
7. Autoclave pouches.
8. Isothermal heating pad (or other heat source).
9. 3-ml syringe with needle, sterile.
10. Anesthetic machine for small animals equipped with oxygen
tank/regulator and isoflurane vaporizer (or other method of
anesthesia).
11. Dissecting microscope.
12. Light source (gooseneck microscope illuminator).
16 Traci A. Wilgus
13. Absorbent pads.

14. Suture (6-0 polypropylene, nonabsorbable suture with C-1
taper needle, or similar).
15. Clippers.
16. Surgical personal protective equipment (gown, mask, hair
bonnet).
17. Sterile surgical gloves.
18. Cotton tip applicators.
2.3 Reagents 1. Sterile saline (0.9% sodium chloride, for injection).

2. India ink.
3. Isoflurane.
4. Chlorhexidine scrub (4% w/v).
5. 70% ethanol.
6. Xylazine.
3 Methods
Before any studies using mice are performed, the institutional

animal care and use committee should be consulted. Official
approval of the protocols and procedures should be obtained
prior to conducting studies.
3.1 Generation 1. Prior to breeding, arrange mice so that they are housed four
of Timed mice per cage for females and one mouse per cage for males.
Pregnant Mice The number of cages will depend on the number of wounded
fetuses desired. Mice should be a minimum of 8–10 weeks of
age for optimal breeding results.
2. Once per week, place female mice from a single cage into the
cage of a designated male. The following morning, check each
female mouse for the presence of a vaginal plug, record the
results, and return the female mice to their original cage (see
Note 1). The day of plug detection is designated E0, which
should be used to calculate the appropriate dates for surgery
(E15 or E18 for scarless or fibrotic healing, respectively) (see
Note 2). Repeat this mating procedure weekly on the same day
of the week, until pregnant mice are obtained.
3. At least weekly, check females for signs of pregnancy. This is
usually obvious based on weight gain and the appearance of a
bulging abdomen. Palpation can also be used if needed. For
surgeries being performed at E15 or older, typically visual signs
of pregnancy are apparent several days prior to the surgery date.
4. At the desired embryonic age, prepare pregnant female mice

for surgery as described below. Continue mating any remaining
female mice that are not pregnant as described above.
3.2 Presurgical 1. Prior to surgeries, sterilize surgical instruments and other

Preparation objects/reagents that will be used during surgery.
2. If an isothermal pad will be used as a heat source, preheat the
pad by microwaving or submerging in a heated water bath as
directed by the manufacturer.
3. Dilute India ink to 10% in sterile saline and dilute or prepare
xylazine and other drugs/experimental reagents that will be
used prior to, during, or after surgery. To maintain sterility, this
should be performed in a biosafety hood. Load the Hamilton
syringe with diluted India ink (see Note 3). Using aseptic
technique, fill several 3-ml syringes with sterile saline (these
will be used to keep the abdomen hydrated during surgery).
4. If inhaled isoflurane will be used for anesthesia, prepare the
anesthetic machine. Fill the anesthetic chamber in the vaporizer
with isoflurane. Turn on the oxygen tank to ensure that there
are sufficient oxygen levels to last for the duration of the
surgery.
5. Prepare the surgical space. Arrange the dissecting scope, light
source, and anesthetic machine appropriately. Disinfect sur-
faces with 70% ethanol. Place a layer of absorbent padding on
the surgical surface, followed by the preheated isothermal pad,
then another layer of absorbent padding.
6. Prepare the animal for surgery. Induce and then maintain
anesthesia with a suitable level of isoflurane. Appropriate
depth of anesthesia should be confirmed by lack of a
toe-pinch response. Using the lowest dose of isoflurane that
will maintain appropriate anesthesia depth will minimize the
risk of complications. Shave the abdomen of the mouse, then
move the shaved mouse to the surgical surface, arranging the
animal in the middle of the warm isothermal pad. After shaving
the mouse, the surgeon should wear a gown, face mask, and
hair bonnet. Disinfect the abdominal skin with chlorhexidine
scrub followed by 70% ethanol using soaked cotton tip appli-
cators. Use a circular motion starting at the middle of the
abdomen and moving outward. Repeat the chlorhexidine and
ethanol washes three times.
7. Open a packet of surgical gloves, sutures, and surgical pack,
which should contain sterilized instruments and gauze. Avoid
contacting the inner contents with nonsterile items from this
point forward. The sterile 3-ml syringes loaded with saline
should also be added. Carefully, put on sterile surgical gloves
without contaminating the working surfaces of the gloves.
18 Traci A. Wilgus
Arrange four pieces of sterile gauze on the abdomen of the

mouse to create a surgical drape, leaving a rectangular space in
the middle large enough for the laparotomy incision to be
made. Use additional sterile gauze to cover any surfaces that
may need to be touched or adjusted during the surgery (light
source, isoflurane gauge, microscope controls, etc.). Once the
animal is prepared, adjust the light source and scope if
necessary.
3.3 Surgical 1. Perform a midline laparotomy. Using forceps, grasp the

Procedures abdominal skin and create a vertical incision along the midline
using scissors. The incision should be about 4 cm in length. In
a similar manner, create an incision in the abdominal muscle
wall. Toothed forceps can be used. Cutting carefully along the
linea alba reduces the chance of bleeding.
2. With closed curved forceps or a needle holder, expose a portion
of the uterus on one side of the animal (there will be a uterine
segment on each side of the mouse) by scooping the instru-
ment under the uterus and gently lifting. Try to minimize the
amount of the uterus exteriorized. Looking through the dis-
secting microscope, identify an embryo that is situated such
that it can be seen clearly through the scope and is in a position
allowing for easy access to the dorsal skin. In our experience, it
is best to avoid the two fetuses directly adjacent to the cervix on
either side. From this point forward during the surgery, the
exteriorized portion of the uterus should be sprayed periodi-
cally with small volumes of sterile saline to maintain hydration.
3. Taking care to avoid cutting any visible vessels in the uterus,
make a small incision (3–4 mm) through the uterine wall using
microsurgical scissors. Then, cut through the amniotic mem-
branes. This should result in visible leakage of amniotic fluid,
especially at earlier gestational ages.
4. Using fine-tipped forceps, gently grip the dorsal skin in the
middle of the fetus and make a small (approximately 2 mm)
full-thickness incision using microsurgical scissors. Care should
be taken to avoid damaging the underlying tissues.
5. Using the Hamilton syringe, inject 1 μl of 10% India ink
solution subcutaneously at the edges of the wound. This allows
for identification of the wound site after healing has occurred.
Spray the area with saline.
6. Close the incision in the uterus with a horizontal mattress
suture.
7. Repeat the wounding process on several fetuses. Limiting the
number of wounded fetuses to four (two on each side of the
uterus) and minimizing the total time of the surgery reduce the
risk of complications. Once the desired number of fetuses has
been wounded, carefully replace the uterus without introdu-

cing torsion.
8. Close the laparotomy incisions in the pregnant female mouse
using multiple simple interrupted sutures, first for the abdomi-
nal wall and then for the skin (see Note 4).
3.4 Postoperative 1. Immediately after surgery, inject xylazine (3 mg/kg) subcuta-

Care and Tissue neously for sedation and analgesia or administer other postop-
Harvest erative analgesics.
2. Return the mouse to its cage, laying it on gauze. Place the
isothermal pad under the cage. Monitor the mouse every
30 min for the first 3 h. Then, remove the gauze from the
cage and return the cage to its housing location. Monitor the
mouse at least once per day until wounds are harvested.
3. At the desired time point post-injury, euthanize the female
mouse in accordance with American Veterinary Medical Asso-
ciation Guidelines. Depending on the postsurgical time point
and the embryonic age at which the surgeries were performed,
wounds need to be harvested either from unborn embryos or
from recently delivered pups. In either case, decapitate the
embryos or postnatal mice with scissors prior to harvesting
skin samples (see Note 5).
4 Notes
1. Since the presence (or absence) of a vaginal plug does not

always accurately predict pregnancy, we separate the female
and male mice after overnight mating and limit mating to
once per week. If natural breeding is not effectively resulting
in pregnancies or if a large number of pregnant mice are
needed, superovulation may be considered [13].
2. In our experience, it is difficult to create wounds on embryos
younger than E15 because the skin is less developed, gelati-
nous, and not firm enough to manipulate with forceps or create
a well-defined incision.
3. Mediators (lipids, recombinant proteins, chemicals, etc.) can
be added to the India ink solution and injected into E15 or E18
wounds to evaluate pro- or anti-fibrotic effects, respectively.
4. Closing the abdominal wall and skin with multiple interrupted
sutures rather than one running suture minimizes the risk of
the incisions reopening if the mouse chews through the suture
material.
5. For harvesting wound tissue at early time points post-
wounding for early embryonic wounds (i.e., E15), the skin is
quite thin and fragile. This can make dissecting the skin/
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you also shall die and rise to life again.’ ” The Hare went to the Men,
and said, “Like as I die and do not rise to life again, so you shall also
die, and not rise to life again.” When he returned, the Moon asked
him, “What hast thou said?” “I have told them, ‘Like as I die and do
not rise to life again, so you shall also die and not rise to life again.’ ”
“What,” said the Moon, “hast thou said that?” And she took a stick
and beat the Hare on his mouth, which was slit by the blow. The
Hare fled, and is still fleeing. 1 [104]
[Contents]
33. A THIRD VERSION OF THE SAME FABLE.

(From an original Manuscript in English, by Mr. John Priestley, in Sir G. Grey’s
Library.)
The Moon, on one occasion, sent the Hare to the earth to inform
Men that as she (the Moon) died away and rose again, so mankind
should die and rise again. Instead, however, of delivering this
message as given, the Hare, either out of forgetfulness or malice,
told mankind that as the Moon rose and died away, so Man should
die and rise no more. The Hare, having returned to the Moon, was
questioned as to the message delivered, and the Moon, having
heard the true state of the case, became so enraged with him that
she took up a hatchet to split his head; falling short, however, of that,
the hatchet fell upon the upper lip of the Hare, and cut it severely.
Hence it is that we see the “Hare-lip.” The Hare, being duly incensed
at having received such treatment, raised his claws, and scratched
the Moon’s face; and the dark parts which we now see on the
surface of the Moon are the scars which she received on that
occasion. [105]
[Contents]
34. A FOURTH VERSION OF THE SAME FABLE.

(From Sir James E. Alexander’s “Expedition of Discovery into the Interior of
Africa,” vol. i. p. 169.)
The Moon, they say, wished to send a message to Men, and the
Hare said that he would take it. “Run, then,” said the Moon, “and tell
Men that as I die and am renewed, so shall they also be renewed.”
But the Hare deceived Men, and said, “As I die and perish, so shall
you also.” 2 [106]
[Contents]
35. A ZULU VERSION OF THE LEGEND OF THE

“ORIGIN OF DEATH.”
(From Manuscript, “Zulu Legends,” No. 214 of Sir G. Grey’s Library, vol. i. part i. p.
107.)
God (Unknlunkuln) arose from beneath (the seat of the spiritual

world, according to the Zulu idea), and created in the beginning 3
men, animals, and all things. He then sent for the Chameleon, and
said, “Go, Chameleon, and tell Men that they shall not die.” The
Chameleon went, but it walked slowly, and loitered on the way,
eating of a shrub called Bukwebezane.
When it had been away some time, God sent the Salamander after
it, ordering him to make haste and tell Men that they should die. The
Salamander went on his way with this message, outran the
Chameleon, and, arriving first where the Men were, told them that
they must die. [107]
1 “We are now angry with the Hare,” say the old Namaqua, “because he brought
such a bad message, and therefore we dislike to eat his flesh.”—Knudsen. ↑
2 Old Namaquas will not therefore touch Hare’s flesh; but the young men may
partake of it; that is, before the ceremony of making them men is performed,
which merely consists in slaughtering and eating an ox or a couple of sheep.—
Alexander. ↑
3 Ohlangeni. Vide Colenso’s “Zulu-English Dictionary,” p. 179. ↑
[Contents]
VII.
HEITSI EIBIP AND OTHER LEGENDS.
[Contents]
36. HEITSI EIBIP.

(From a German original Manuscript in Sir G. Grey’s Library, H. C. Knudsen’s
“Notes on the Hottentots,” p. 7.)
Heitsi Eibip, or Kabip, was a great and celebrated sorcerer among

the Namaqua. He could tell secret things, and prophesy what was to
happen afterwards.
Once he was travelling with a great number of people, and an enemy

pursued them. On arriving at some water he said, “My grandfather’s
father, open thyself that I may pass through, and close thyself
afterwards.” So it took place as he had said, and they went safely
through. Then their enemies tried to pass through the opening also,
but when they were in the midst of it, it closed again upon them, and
they perished. 1 [108]
Heitsi Kabip died several times, and came to life again. When the
Hottentots pass one of his graves they throw a stone on it for good
luck. 2
Heitsi Eibip could take many different forms. Sometimes he

appeared handsome, very handsome, or his hair grew long down to
his shoulders; at other times it was again short. [109]
[Contents]
37. THE VICTORY OF HEITSI EIBIP.

“Notes on the Hottentots,” p. 7.)
At first they were two. One had made a large hole in the ground, and
sat by it, and told passers-by to throw a stone at his forehead. The
stone, however, rebounded and killed the person who had thrown it,
so that he fell into the hole. At last Heitsi Eibip was told that in this
manner many people died. So he arose and went to the man, who
challenged Heitsi Eibip to throw (a stone) at him. The latter, however,
declined, for he was too prudent; but he drew the man’s attention to
something on one side, and while he turned round to look at it, Heitsi
Eibip hit him behind the ear, so that he died and fell into his own
hole. After that there was peace, and people lived happily. 3 [110]
[Contents]
38. ANOTHER VERSION OF THE SAME LEGEND.

(The original, in the Hottentot language, is in Sir G. Grey’s Library, G. Krönlein’s
Manuscript, p. 36.)
All men who came near to that hole were, it is said, pushed down
into it by the ǂGã ǂgorip 4 (the pusher into the hole), as he knew well
where it lay. Whilst he was thus employed, there came the Heitsi
Eibip, called also Heigeip, and saw how the ǂGã ǂgorip treated the
people.
Then these two began to hunt each other round the hole, saying—
“Push the Heigeip down!”

“Push the ǂGã ǂgorip down!”
With these words they hunted each other round for some time; but at
last the Heigeip was pushed [111]down. Then he said to the hole,
“Support me a little,” and it did. Thus, being supported, he came out;
and they hunted each other again with the same words:—

A second time the Heigeip was pushed down, and he spoke the
same words: “Support me a little,” and thus got out again.
Once more these two hunted after each other, till at last the ǂGã
ǂgorip was pushed down, and he came not up again. Since that day
men breathed freely and had rest from their enemy, because he was
vanquished. [112]
[Contents]
39. THE RAISIN-EATER.

Manuscript, pp. 34, 35.)
It is said that when Heitsi Eibip was travelling about with his family,
they came to a valley in which the raisin-tree was ripe, and he was
there attacked by a severe illness. Then his young (second) wife
said, “This brave one is taken ill on account of these raisins; death is
here at the place.” The old man (Heitsi Eibip), however, told his son
ǃUrisip 5 (the whitish one), “I shall not live, I feel it; thou must,
therefore, cover me when I am dead with soft stones.” And he spoke
further, “This is the thing which I order you to do:—‘Of the raisin-
trees of this valley ye shall not eat. For if ye eat of them I shall infect
you, and ye will surely die in a similar way.’ ”
His young wife said, “He is taken ill on account of the raisins of this
valley. Let us bury him quickly, and let us go.” [113]
So he died there, and was covered flatly with soft stones according
as he had commanded. Then they went away from him.
When they had moved to another place, and were unpacking there,
they heard always from the side whence they came a noise as of
people eating raisins and singing. In this manner the eating and
singing ran:—
“I, father of ǃUrisip,

Father of this unclean one,
I, who had to eat these raisins, and died,
And dying live.”
The young wife perceived that the noise came from the side where
the old man’s grave was, and said, “ǃUrisip! Go and look!” Then the
son went to the old man’s grave, where he saw traces which he
recognised to be his father’s footmarks, and returned home. Then
the young wife said, “It is he alone; therefore act thus:—
“Do so to the man who ate raisins on the windward side,

Take care of the wind that thou creepest upon him from the leeward;
Then intercept him on his way to the grave, [114]
And when thou hast caught him, do not let him go.”
He did accordingly, and they came between the grave and Heitsi
Eibip who, when he saw this, jumped down from the raisin-trees, and
ran quickly, but was caught at the grave. Then he said:
“Let me go! For I am a man who has been dead that I may not infect
you!” But the young wife said, “Keep hold of the rogue!” So they
brought him home, and from that day he was fresh and hale. [115]
[Contents]
40. ORIGIN OF THE DIFFERENCE IN MODES OF

LIFE BETWEEN HOTTENTOTS AND BUSHMEN.
“Notes on the Hottentots,” pp. 7, 8.)
In the beginning there were two. One was blind, the other was
always hunting. This hunter found at last a hole in the earth, from
which game proceeded, and killed the young. The blind man, feeling
and smelling them, said, “They are not game, but cattle.”
The blind man afterwards recovered his sight, and going with the
hunter to this hole, saw that they were cows with their calves. He
then quickly built a kraal (fence made of thorns) round them, and
anointed himself, just as Hottentots (in their native state) are still
wont to do.
When the other, who now with great trouble had to seek his game,
came and saw this, he wanted to anoint himself also. “Look here!”
said the other, “you must throw the ointment into the fire, and
afterwards use it.” He followed this advice, and the flames [116]flaring
up into his face, burnt him most miserably; so that he was glad to
make his escape. The other, however, called to him: “Here, take the
kirri (a knobstick), and run to the hills, to hunt there for honey.”
Hence sprung the race of Bushmen. [117]
1 Knudsen, who heard this legend from the Hottentot Petrus Friedrik, was
afterwards informed that Heitsi Eibip [108]was not the person meant in this tale.
It looks very much like the end of our 27th Fable, of the Woman who outwitted the
Elephants. ↑
2 Sir James E. Alexander, in his “Expedition of Discovery into the Interior of
Africa,” vol. i. p. 166, speaking of the people at Warm Bath, or Nisbett Bath,
says:—“These Namaquas thought that they came from the East. In the country
there is occasionally found (besides the common graves covered with a heap of
stones) large heaps of stones, on which had been thrown a few bushes; and if the
Namaquas are asked what these are, they say that Heije Eibib, their Great Father,
is below the heap; they do not know what he is like, or what he does; they only
imagine that he also came from the East, and had plenty of sheep and goats; and
when they add a stone or branch to the heap, they mutter, ‘Give us plenty of
cattle.’ ” ↑
3 Sir James Alexander, in his “Expedition of Discovery into the Interior of Africa,”
vol. ii. p. 250, states:—“On the 3rd of August the waggon went on to Aneip, or
Wet Foot, and I went out of the way with Jan Buys, and two or three men, to see a
hole, which was supposed to be inhabited by Heije Eibib, and was the wonder of
the country.” ↑
4 The ǂ is the palatal click, described in Note to Fable 24, p. 55; and indicates the
nasal pronunciation of a syllable. ↑
5 The ǃ is the cerebral click described in Note to Fable 27, p. 62. ↑
[Contents]
VIII.
HOUSEHOLD TALES.
[Contents]
41. THE LITTLE WISE WOMAN.

Manuscript, p. 53.)
A girl, it is said, went to seek for onions. As she arrived at the place
where they grew, she met with some men, one of whom was blind
(i.e., half-blind, having only one eye). As she dug (for the onions) the
men helped her, digging also. When her sack was full, they said to
her, “Go, tell the other girls, that many of you may come.” So she
went home and told her companions, and early the next morning
they started. But a little girl followed them. The other girls said, “Let
the little girl go back.” But her elder sister protested against this,
saying, “She runs by herself, you need not put her into your awa-
skin.”
So they went all together, and having reached the onion-ground,

began to dig. Now the little girl saw [118]traces of feet, and said to the
one who had guided them thither, “Wonderful! whence so many
traces? Were you not alone here?” The other replied, “I walked
about and looked out; therefore they must of course be many.” The
child, however, did not believe that if the other girl had been alone
the traces could be many, and felt uneasy; for she was a wise little
woman. From time to time she rose (from her work) and peeped
about, and once, while doing this, found by chance an ant-eater’s
hole. Still further spying about, she perceived some men, but they
did not see her. She then returned and continued digging with the
other girls, without, however, saying anything. But in the midst of
their work she always rose and looked about her. So the others
asked her, “Why do you always spy about you, and leave off
digging? What a girl!” But she continued her work in silence. When
she rose from it again, she saw the men approaching. As they drew
near the One-eyed blew through a reed pipe the following air:—
“To-day there shall blood flow, blood flow, blood flow!”
The little girl understood what was blown on the reed. She said to
the elder ones, whilst they were dancing, “Do you also understand
the tune that is blown on the reed?” But they only said, “What a
[119]child she is!” So she mixed in the dance with the others; but
managed while so doing to tie her sister’s caross-cloak to her own,
and in this manner they danced on, till it became very noisy, and
then they found an opportunity to slip away.
On their way out the little sister asked, “Do you understand the reed
—I mean what is blown on it?” She answered, “I do not understand
it.” Then the little girl explained to her that the tune on the reed said,
“To-day blood shall flow!” When they walked along, the little girl let
her elder sister go first, and herself followed, walking backwards, and
carefully stepping in her sister’s traces, so that they thus left only one
set of footmarks, and these going in a contrary direction. In this
manner they arrived at the ant-eater’s hole.
But the men killed all those girls who had remained dancing with
them. When the eldest of those who had escaped heard their
wailing, she said, “Alas, my sisters!” But the younger one answered
her, “Do you think you would have lived if you had remained there?”
Now “One-eye” was the first to miss the sisters, and said to the other
men, “Where may the two handsome girls be who danced with me?”
The others replied, “He lies. He has seen with his eye” (satirically
[120]meaning he had seen wrongly). But “One-eye” insisted that “two
girls were truly missing.” Then they went to find their spoor, but the
traces had been rendered indistinct enough to puzzle them.
When the men arrived at the ant-eater’s hole, they could not see that
the footmarks went further, so they spied into the hole, but saw
nothing. Then “One-eye” looked also, and he saw the girls, and
cried, “There they sit.” The others now looked again, but still saw
nothing; for the girls had covered themselves with cobwebs.
One of the men then took an assegai, and piercing through the
upper part of the hole, hit the heel of the larger girl. But the little wise
woman took hold of the assegai, and wiped off the blood. The elder
sister was about to cry, but the little one warned her not.
When “One-eye” spied again, the little girl made big eyes at him. He
said, “There she sits.” The others looked too, but as they could see
nothing they said (satirically), “He has only seen with his eye.”
At last the men got thirsty, and said to “One-eye,” “Stay you here,
and let us go to drink, and when we have returned you may go also.”
When “One-eye” was left alone there, the little girl said (conjuring
him): [121]
“You dirty son of your father,

Are you there? Are you alone not thirsty?
Oh, you dirty child of your father!
Dirty child of your father!”
“I am indeed thirsty,” said “One-eye,” and went away.
Then the two girls came out of the hole, and the younger one took
her elder sister on her back, and walked on. As they were going over
the bare, treeless plain, the men saw them, and said, “There they
are, far off,” and ran after them.
When they came near, the two girls turned themselves into thorn
trees, called “Wait-a-bit,” and the beads which they wore became
gum on the trees. The men then ate of the gum and fell asleep.
Whilst they slept, the girls smeared gum over the men’s eyes and
went away, leaving them lying in the sun.
The girls were already near their kraal, when “One-eye” awoke, and
said:
“Oh, the disgrace! fie on thee!

Our eyes are smeared over; fie on thee, my brother!”
Then they removed the gum from their eyes, and hunted after the
girls; but the latter reached home in safety, and told their parents
what had happened.
Then all lamented greatly, but they remained quietly at home, and
did not search for the other girls. [122]
[Contents]
42. THE UNREASONABLE CHILD TO WHOM THE

DOG GAVE ITS DESERTS;
OR, A RECEIPT FOR PUTTING ANY ONE TO

SLEEP.
(The original, in the o Tyi-hereró or Dámara language, is in Sir G. Grey’s Library, J.
Rath’s Manuscript, pp. 39, 43.)
There was a little girl who had an eïngi (pronounced a-inghi, some
kind of fruit). She said to her Mother, “Mother, why is it that you do
not say, ‘My first-born, give me the eïngi?’ Do I refuse it?”
Her Mother said, “My first-born, give me the eïngi.” She gave it to her
and went away, and her Mother ate the eïngi.
When the child came back, she said, “Mother, give me my eïngi?”
but her Mother answered, “I have eaten the eïngi!”
The child said, “Mother, how is it that you have eaten my eïngi, which
I plucked from our tree?” The Mother then (to appease her) gave her
a needle.
The little girl went away and found her Father sewing thongs with
thorns; so she said, “Father, how is it that you sew with thorns? Why
do not you say, [123]‘My first-born, give me your needle?’ Do I
refuse?” So her Father said, “My first-born, give me your needle.”
She gave it to him and went away for a while. Her Father
commenced sewing, but the needle broke; when, therefore, the child
came back and said, “Father, give me my needle,” he answered,
“The needle is broken;” but she complained about it, saying, “Father,
how is it that you break my needle, which I got from Mother, who ate
my eïngi, which I had plucked from our tree?” Her Father then gave
her an axe.
Going farther on she met the lads who were in charge of the cattle.
They were busy taking out honey, and in order to get at it they were
obliged to cut down the trees with stones. She addressed them: “Our
sons, how is it that you use stones in order to get at the honey? Why
do not you say, ‘Our first-born, give us the axe?’ Do I refuse, or what
do I?” They said, “Our first-born, give us the axe.” So she gave it
them, and went away for some time. The axe broke entirely. When
she came back she asked, “Where is the axe? Please give it me.”
They answered, “The axe is broken.” She then said, “How is it that
you break my axe, which I had received from Father who had broken
my needle, which I got from Mother who had eaten my eïngi, which I
[124]had plucked from our tree?” But they gave her some honey (to
comfort her).
She went her way again, and met a little old woman, eating insects,
to whom she said, “Little old woman, how is it that you eat insects?
Why don’t you say, ‘My first-born, give me honey?’ Do I refuse or
not?” Then the little old woman asked, “My first-born, give me
honey.” She gave it her and went away; but presently returning, said,
“Little old woman, let me have my honey!” Now the old woman had
managed to eat it all during her absence, so she answered, “Oh! I
have eaten the honey!” So the child complained, saying, “How is it
that you eat my honey, which I received from the lads of our cattle,
from our children who had broken my axe, which had been given me
by Father who had broken my needle, which was a present from my
Mother who had eaten up my eïngi, that I had plucked from our
tree?”
The little old woman gave her food, and she went away. This time
she came to the pheasants, who scratched the ground; and she
said, “Pheasants! how is it that you scratch the ground? Why do not
you say, ‘First-born, give us food?’ Do I refuse, or what do I?” They
said, “First-born, give.” So she gave to them, and went away. When
she came [125]back and demanded her food again, they said, “We
have eaten the food.” She asked, “How is it that you eat my food,
which I had received from a little old woman who had eaten up my
honey, that I had got from the lads of our cattle who had broken my
axe, which had been given me by my Father who had broken my
needle, which was a present from my Mother who had eaten my
eïngi, which I had plucked from our tree?” The pheasants, flying up,
pulled out each one a feather and threw them down to the little girl.
She then, walking along, met the children who watched the sheep.
They were plucking out hairs from the sheep-skins. So she asked
them, “How is it that you pull at these skins? Why do not you say,
‘First-born, give us the feathers?’ Do I refuse, or what do I?” They
said, “First-born, give us the feathers.” She gave them and went
away, but all the feathers broke. When she returned and said, “Give
me my feathers,” they answered, “The feathers are broken.” Then
she complained, “Do you break my feathers which I received from
the pheasants who had eaten my food, which had been given me by
a little old woman?” They gave her some milk.
She went again on her way, and found their own [126]handsome dog
gnawing bones. She said, “Our dog, how is it that you gnaw these
bones?” The dog answered, “Give me milk.” She gave it him, and he
drank it all. Then she said to the dog, “Give me back my milk.” He
said, “I drank it.” She then repeated the same words which she had
spoken so often before; but the dog ran away, and when she
pursued him, he scampered up a tree. She climbed up after him, but
the dog jumped down again on the other side. She wanted to do the
same, but could not. Then she said, “Our dog, please help me
down.” He answered, “Why did you pursue me?” and ran away
leaving her up the tree.
“That is enough,” say the Damara. [127]

[Contents]
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Amarillo, TX, USA Hiranmoy Das

1 Investigating Epidermal Interactions Through an In Vivo Cutaneous

13 Electrospun Aligned Coaxial Nanofibrous Scaffold for Cardiac Repair . . . . . . . . . 129

VINAY KUMAR AAKALU • Department of Ophthalmology and Visual Sciences, University of

DANIELA ROLPH • Department of Pharmaceutical Sciences, Jerry H. Hodge School of

Investigating Epidermal Interactions Through an In Vivo

Key words Wound healing, Microenvironment, Epidermal

Cutaneous wound healing is an intricate and multifaceted process.

One of the most robust elements of this assay resides in the

[7]. Non-healing wounds, chronic ulcers, hypertrophic scars,

Animal experiments should take place according to the Institu-

2.1 Preparation of 1. Isoflurane or other inhalation anesthetic.

5. Scalpel #11 blade, one per three animals to be wounded (see

2.3 Wound 1. Isoflurane or other inhalation anesthetic.

2.4 Analysis of 1. Graphing or statistical software package (see Note 2).

2.5 Preparation of 1. Materials for appropriate euthanasia.

3.2 Wound Incision 1. Approximately 24 h after shaving, briefly anesthetize animals

Fig. 1 Inspecting animals for injury post-shaving. Animals showing signs of

6. Holding a scalpel vertically, the second investigator should

3.3 Wound 1. Carefully anesthetize the animals (see Note 14).

Fig. 4 Measurement of incisional wounds. Wounds can take on asymmetrical

3. Record when the wound is closed (see Note 16).

1. The scalpel type depends largely on the preference of the

significant differences influenced by genetic background, par-

A Murine Incisional Fetal Wound-Healing Model to Study

Key words Scarless, Regeneration, Wound healing, Fetal, Skin, Development

The repair of wounds in developed skin is an intricate process that

While scar tissue is a concern with healing in mature skin, fetal

2.1 Animals 1. FVB mice (or similar strain).

2.2 Surgical 1. Forceps (fine-tipped, toothed, curved, and blunt-end forceps

13. Absorbent pads.

2.3 Reagents 1. Sterile saline (0.9% sodium chloride, for injection).

Before any studies using mice are performed, the institutional

4. At the desired embryonic age, prepare pregnant female mice

3.2 Presurgical 1. Prior to surgeries, sterilize surgical instruments and other

Arrange four pieces of sterile gauze on the abdomen of the

3.3 Surgical 1. Perform a midline laparotomy. Using forceps, grasp the

been wounded, carefully replace the uterus without introdu-

3.4 Postoperative 1. Immediately after surgery, inject xylazine (3 mg/kg) subcuta-

1. Since the presence (or absence) of a vaginal plug does not

33. A THIRD VERSION OF THE SAME FABLE.

34. A FOURTH VERSION OF THE SAME FABLE.

35. A ZULU VERSION OF THE LEGEND OF THE

God (Unknlunkuln) arose from beneath (the seat of the spiritual