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Rheumatoid Arthritis Methods and Protocols Methods in Molecular Biology 2766 2nd Edition Shuang Liu
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Synthetic Biology Methods and Protocols Methods in Molecular Biology 2760 2nd Edition Braman Jeffrey Carl Edt
University of Hertfordshire Hatfield, Hertfordshire, UK
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RheumatoidArthritis
Methods and Protocols
Second Edition
Edited by Shuang Liu
Department of Pharmacology, Ehime University School of Medicine, Toon, Ehime, Japan
Editor
Shuang Liu Department of Pharmacology
Ehime University School of
Medicine
Toon, Ehime, Japan
ISSN 1064-3745ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-0716-3681-7ISBN 978-1-0716-3682-4 (eBook) https://doi.org/10.1007/978-1-0716-3682-4
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Preface
Welcome to the second edition of Rheumatoid Arthritis: Methods and Protocols. This comprehensive volume represents a testament to the ever-evolving field of rheumatoid arthritis (RA) research, where groundbreaking advances continue to emerge on multiple fronts. As we embark on this journey through the labyrinthine world of RA investigation, we find ourselves at the forefront of a scientific endeavor that holds the promise of transforming the lives of millions afflicted by this debilitating autoimmune disease.
Rheumatoid arthritis is a complex, systemic autoimmune disorder characterized by chronic inflammation, joint destruction, and a wide spectrum of associated comorbidities. Despite the significant progress made in understanding the pathogenesis and treatment of RA in recent years, there is an unceasing need for innovative approaches, methodologies, and therapies to further enhance our understanding and management of this disease.
This second edition builds upon the foundations laid by its predecessor, expanding and refining the content to encompass the most cutting-edge laboratory and clinical protocols employed in RA research today. The book’s contents have been meticulously curated to provide a comprehensive roadmap for researchers, clinicians, and students who aspire to delve deep into the intricate world of rheumatoid arthritis.
In this edition, we examine the multifaceted aspects of RA research, covering a wide range of methodologies and techniques. We explore the utilization of cell culture systems to decipher the intricate molecular pathways underlying RA pathogenesis. We navigate through the intricate realm of animal models that mirror the disease’s complexity and provide invaluable insights for translational research. Genetic modification techniques unveil the genetic underpinnings of RA susceptibility and offer potential avenues for therapeutic intervention.
Furthermore, we delve into the development of novel therapeutics, including the promising realms of aptamers and antibodies. In silico docking and bioinformatics methods offer a computational approach to drug discovery, saving time and resources in the quest for effective treatments. We dive deep into the world of RNA sequencing, exploring bioinformatics techniques to unlock the secrets hidden within the vast transcriptomic landscape of RA.
Finally, we navigate the challenging waters of clinical research, where the knowledge gleaned from laboratories meets the real-world complexities of patient care. We explore the latest clinical study designs, patient-centered approaches, and emerging trends in RA management.
This second edition of Rheumatoid Arthritis: Methods and Protocols stands as a testament to the dedication and collaborative efforts of researchers worldwide. It is our hope that this book will serve as an indispensable resource, guiding you through the ever-evolving landscape of RA research and facilitating breakthroughs that will ultimately improve the lives of individuals living with this chronic condition.
We extend our heartfelt gratitude to the authors, contributors, and the scientific community for their unwavering commitment to advancing our understanding of rheumatoid arthritis. May this book inspire and empower the next generation of researchers to continue pushing the boundaries of knowledge and innovation in the quest to conquer this challenging autoimmune disease.
Toon, Ehime, JapanShuang Liu
Yasuyuki Suzuki
Noritaka Saeki and Akihiro Nakata
Yasuyuki Suzuki and Shuang Liu
33 Assessment of Disease Activity, Structural Damage, and Function in Rheumatoid Arthritis
Jun Ishizaki and Hitoshi Hasegawa
34 Assessment of Musculoskeletal Ultrasound of Rheumatoid Arthritis. .
Jun Ishizaki
35 16S rRNA Gene Amplicon Analysis of Human Gut Microbiota. .
Noriyuki Miyaue
Index
Contributors
HITOSHI HASEGAWA • Department of Hematology, Clinical Immunology, and Infectious Diseases, Ehime University Graduate School of Medicine, Toon, Ehime, Japan
MAKOTO INUI • Department of Pharmacology, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi, Japan
JUN ISHIZAKI • Department of Hematology, Clinical Immunology and Infectious Diseases, Ehime University Graduate School of Medicine, Toon, Ehime, Japan
TAKESHI KIYOI • Division of Analytical Bio-medicine, Department of Pharmacology, Kanazawa Medical University, Kahoku, Japan
SHUANG LIU • Department of Pharmacology, Ehime University Graduate School of Medicine, Toon, Ehime, Japan
MOCHITSUKI MARII • Department of Pharmacology, Ehime University Graduate School of Medicine, Toon, Ehime, Japan
SAHO MARUYAMA • Department of Basic Medical Research and Education, Ehime University Graduate School of Medicine, Toon, Ehime, Japan
NORIYUKI MIYAUE • Department of Clinical Pharmacology and Therapeutics, Ehime University Graduate School of Medicine, Toon, Ehime, Japan
MAYA MIYOSHI • Department of Pharmacology, Ehime University Graduate School of Medicine, Toon, Ehime, Japan
MASAKI MOGI • Department of Pharmacology, Ehime University Graduate School of Medicine, Toon, Ehime, Japan
AKIHIRO NAKATA • Department of Pathophysiology, Ehime University Graduate School of Medicine, Toon, Ehime, Japan
NORITAKA SAEKI • Division of Medical Research Support, Advanced Research Support Center, Ehime University, Toon, Ehime, Japan; Division of Integrative Pathophysiology, ProteoScience Center, Ehime University, Toon, Ehime, Japan
YASUYUKI SUZUKI • Department of Anaesthesiology, Saiseikai Matsuyama Hospital Matsuyama, Ehime, Japan; Department of Pharmacology, Ehime University Graduate School of Medicine, Toon, Ehime, Japan; Research Division, Saiseikai Research Institute of Health Care and Welfare, Tokyo, Japan
HIROYUKI TAKEDA • Proteo-Science Center, Ehime University Matsuyama, Ehime, Japan
ERIKA TAKEMASA • Department of Pharmacology, Ehime University Graduate School of Medicine, Toon, Ehime, Japan
WEI ZHOU • Proteo-Science Center, Ehime University Matsuyama, Ehime, Japan
Part I
Animal Models
Chapter 1
Collagen-Induced Arthritis Models
Maya Miyoshi and Shuang Liu
Abstract
Due to the limitations of using patient-derived samples for systemic kinetic studies in rheumatoid arthritis (RA) research, animal models are helpful for further understanding the pathophysiology of RA and seeking potential therapeutic targets or strategies. The collagen-induced arthritis (CIA) model is one of the standard RA models used in preclinical research. The CIA model shares several pathological features with RA, such as breach of tolerance and generation of autoantibodies targeting collagen, synovial inflammatory cell infiltration, synovial hyperplasia, cartilage destruction, and bone erosion. In this chapter, a protocol for the successful induction of CIA in mice is described. In this protocol, CIA is induced by active immunization by inoculation with type II heterologous collagen in Freund’s adjuvant in susceptible DBA/1 mice.
Key words Collagen-induced arthritis, Freund’s adjuvant, Type II collagen, Emulsion, Immunization
1 Introduction
Rheumatoid arthritis (RA) is a chronic inflammatory disease that initially affects the joints, manifesting as pain, stiffness, and synovitis, leading to cartilage and bone erosion by invading fibrovascular tissue [1]. The central pathogenesis of RA is characterized by the activation of macrophages by autoreactive T cells, resulting in the release of a series of proinflammatory cytokines. However, how the systemic chronic inflammatory state triggers the onset of articular disorder is still poorly understood [2]. To further define the pathogenesis of RA, it is helpful to study human-derived cells and explanted tissues from patients who have undergone arthroscopic surgery or prosthetic replacement arthroplasty. However, this has significant limitations for systemic kinetic studies. Therefore, animal models are not only essential to facilitate understanding of the pathophysiology of RA and seek potential therapeutic targets or strategies but are also the starting point for in vivo application of new therapeutic agents.
Based on the methods of induction, systemically induced models include those elicited by active immunization, such as
collagen-induced arthritis model and proteoglycan-induced arthritis model, those elicited by passive immunization, such as collagen antibody-induced arthritis model and K/BxN antibodyinduced arthritis model, and those elicited by administration of irritant chemicals resulting in chronic inflammation [1, 3]. Each animal model is only an experimental tool that mimics a part of the disease and cannot reproduce the entire condition of human RA. The choice of model depends on the phase of the disease to be studied and the question to be addressed.
The collagen-induced arthritis (CIA) model is a long-lasting and well-explored mouse model for RA. CIA and RA initial similarity in breach of tolerance and generation of autoantibodies targeting collagen, one of the important self-antigens that are also observed in human RA [2, 4]. CIA is induced by active immunization by inoculation with type II heterologous collagen (CII) in Freund’s adjuvant in susceptible strains of mice. DBA/1 mice are commonly used for the CIA model. The model requires at least 6–8 weeks for the accomplishment of clinical signs of disease, such as polyarthritis characterized by synovial inflammatory cell infiltration, synovial hyperplasia, cartilage destruction, and bone erosion [5, 6]. The autoreactive antibody observed in CIA mice is predominately IgG2 subclass, and high levels of both IgG2a and IgG2b are observed at the peak of CIA. Typical cytokine axes involving in human RA pathogenies, such as proinflammatory type 1 T help (Th1) cell-axis, anti-inflammatory cytokine interleukin (IL)-10 axis, and Th17 cell-axis, can be investigated using the CIA model [6, 7]. These characteristics of the CIA model make it the gold standard in vivo model for RA studies.
In this chapter, a protocol for the successful induction of CIA in mice is described. Like any other antigen-induced model, certain technical skills and stable environmental factors are required. The highest arthritis incidence is obtained if the emulsion is correctly performed using bioactivity-qualified CII and appropriate intradermal immunization is performed.
2 Materials
2.1 Emulsion Preparation
1. Type 2 collagen (2 mg/mL, immunization grade) (see Note 1).
2. Incomplete Freund’s adjuvant (IFA).
3. Complete Freund’s adjuvant (CFA).
4. Glass syringes without needles (1 mL) (Hamilton).
5. Electronic homogenizer with a small blade (diameter of 5 mm or less).
6. T-shape stopcock.
7. 5-mL and 10-mL disposable plastic syringes.
2.2 Animal Immunization
1. DBA/1 mice (male, 8–10 weeks old) (see Note 2).
2. 70% ethanol.
3. CII emulsion (CFA)/CII emulsion (IFA).
4. 25 and 27 gauge × 5/8″ needles.
3 Methods
3.1 Emulsion
Preparation (See Notes 3 and 4)
3.2 Animal
Immunization (See Note 7) (Fig. 1)
1. Fill glass syringes with 500 μL of CFA (IFA for booster injection) and 500 μL of immunization grade CII, respectively.
2. Seal the tips of both syringes with a T-shape stopcock.
3. Connect the rest of the connector of the T-shape stopcock with a 5-mL or 10-mL plastic syringe without a plunger and cut halfway from the plunger opening.
4. Push the plunger of the glass syringes and let CFA (IFA for booster injection) and CII solution mix in the plastic syringe. Air bubbles should be avoided during solution mixing.
5. After sealing the plastic syringe with the T-shape stopcock, take off the glass syringes.
6. Clamp the syringe to a ring stand and place it in an ice water bath to keep the emulsion cool during mixing.
7. Homogenize the mixture to emulsify CFA (IFA for booster injection) with the collagen solution until the emulsion is stable (see Note 5).
8. Transfer the emulsion to a 1-mL glass syringe for animal injection (see Note 6). The prepared emulsion should be injected into animals as soon as possible (within 1 h). Keep the emulsion cool at 4 °C until use.
1. DBA/1 mice are used for induction of CIA. Primary intradermal injection of CII and CFA emulsion is performed at a site 2 cm distal to the base of the tail on day 0.
2. Use a squirt bottle to apply 70% ethanol to the injection site and wipe with tissue.
3. Place a 25-or 27-gauge needle on the glass syringe. Wipe the needle to prevent leakage of emulsion.
4. Inject 100 μL (100 μg CII/ mouse) of CII and CFA emulsion intradermally at the base of the tail, with noticeable tissue resistance to the injection (see Note 8).
5. Put the mouse in a clean cage, and house the mice in specific pathogen-free (SPF) conditions.
Fig. 1 Typical appearances of hindpaws of (a) non-arthritis control mouse and (b) collagen-induced arthritis (CIA) mouse. Erythema and edema are observed in CIA mouse
6. Administer a booster injection of emulsion of CII and IFA on day 21. The injection site is about 3 cm from the base of the tail. Choose a different location from the initial injection site.
7. Use a squirt bottle to apply 70% ethanol to the injection site and wipe with tissue.
8. Place a 25-or 27-gauge needle on the glass syringe. Wipe the needle to prevent leakage of emulsion.
9. Insert the needle 3 cm from the base of the tail until the tip reaches 1.5 cm from the base. Inject 100 μL (100 μg CII/ mouse) of CII and IFA emulsion intradermally at the base of the tail, with noticeable tissue resistance to the injection.
10. Put the mouse in a clean cage and house the mice in SPF conditions. The incidence of CIA should be 90–100% at 42–56 days. The CIA mice are ready for evaluation of arthritis severity.
4 Notes
1. Immunization-grade CII should be solubilized and stored in a diluted solution of acetic acid.
2. DBA/a (H-2q) and B10.RIII(H-2r) are highly susceptible to CIA. DBA/I mice respond to chick, bovine, and porcine type II collagen, while B10.RIII mice respond to bovine and porcine collagen, but poorly respond to chick and human collagen.
3. The procedures for emulsion preparation should be performed under sterile conditions.
4. A method using an electric homogenizer is highly recommended for preparing emulsion. Do not use syringe–syringe or sonication methods in the establishment of CIA.
5. The highest arthritis incidence is obtained if the emulsion is correctly performed so that it has a consistency of dense whipped cream and it should not disperse quickly when a droplet of emulsion is placed on the surface of water.
6. It is sometimes difficult to move the plunger when a plastic disposable syringe is used.
7. The animal experiment protocols should be performed in accordance with the guidelines of the Animal Care Committee of the institute.
8. If the injection is rapid and easy without tissue resistance, it can result in a low incidence of CIA.
References
1. Liu S, Kiyoi T, Takemasa E, Maeyama K (2015) Systemic lentivirus-mediated delivery of short hairpin RNA targeting calcium release-activated calcium channel 3 as gene therapy for collageninduced arthritis. J Immunol 194:76–83
2. McInnes IB, Schett G (2011) The pathogenesis of rheumatoid arthritis. N Engl J Med 365: 2205–2219
3. Bessis N, Decker P, Assier E, Semerano L, Boissier MC (2017) Arthritis models: usefulness and interpretation. Semin Immunopathol 39:469–486
Trentham DE (1982) Collagen arthritis as a relevant model for rheumatoid arthritis. Arthritis Rheum 25:911–916
5. Caplazi P, Baca M, Barck K, Carano RA, DeVoss J, Lee WP et al (2015) Mouse models of rheumatoid arthritis. Vet Pathol 52:819–826
6. Miyoshi M, Liu S, Morizane A, Takemasa E, Suzuki Y, Kiyoi T et al (2018) Efficacy of constant long-term delivery of YM-58483 for the treatment of rheumatoid arthritis. Eur J Pharmacol 824:89–98
7. Mauri C, Williams RO, Walmsley M, Feldmann M (1996) Relationship between Th1/Th2 cytokine patterns and the arthritogenic response in collagen-induced arthritis. Eur J Immunol 26: 1511–1518
Chapter 2
Human Xenograft Model
Shuang Liu
Abstract
Human-SCID grafting is a commonly used technique for the long-term investigation of rheumatoid arthritis (RA) explants. To establish a chimeric immunological system in NOD/SCID mice, RA patientderived pannus tissue from the synovial membrane, articular cartilage, and bone can be transplanted subcutaneously. Same patient-derived peripheral mononuclear cell chimerism can be successfully achieved by intraperitoneal engraftment. This xenograft model is able to be used for the initial screening of human target-specified biologics.
Key words Xenograft rheumatoid arthritis model, NOD/SCID mouse, Peripheral mononuclear cell, Ar ticular tissue, Synovial invasion
1 Introduction
Several animal models, including antigen-induced models, such as collagen-induced arthritis, and spontaneous models, such as TNF-α transgenic mice and SKG mice, have been developed for the study of rheumatoid arthritis (RA). However, these models are not able to be used for in vivo screening of human target-specified biologics, especially for chimeric, humanized, and human-type monoclonal antibody or gene therapeutic products, which have been widely studied for clinical treatment of RA patients. Therefore, a xenograft model, in which human-derived explants are transplanted to a severe combined immunodeficiency (SCID) mouse, has been established for human target-specified biologics screening.
It was first reported that RA synovial tissue could be transplanted into SCID mice, and this animal model was useful for studying the pathogenesis of RA and the development of antirheumatic drugs in the early 1990s [1]. The initial studies were conducted on small pieces of synovium transplanted beneath the renal capsule in the mice. The maintenance of human-derived lymphocytes was poor, and usage of the model was limited. Next,
Shuang Liu (ed.), Rheumatoid Arthritis: Methods and Protocols, Methods in Molecular Biology, vol. 2766, https://doi.org/10.1007/978-1-0716-3682-4_2,
a challenge approach in which transplantation was changed to subcutaneous tissue on the back of SCID was conducted. In this model, tissue with a relatively large size, such as pannus tissue from the synovial membrane, articular cartilage, and bone, collected together from RA patients at the time of prosthetic replacement arthroplasty, was used for transplantation [2, 3]. The histologic features of human RA, such as pannus formation, proliferative synovial fibroblasts, osteoclasts and hyaluronic acid-positive articular cartilage, were observed. Based on the technique of the subcutaneous xenograft model, a chimeric human–mouse model was established using NOD/SCID mice, which are characterized by the absence of functional T cells and B cells, deficient NK function, lymphopenia, hypogammaglobulinemia, and a normal hematopoietic microenvironment. Patient-derived synovial tissue, bone, and articular cartilage were xenografted into NOD/SCID mice. To mimic the supporting inflammatory microenvironment of RA, peripheral blood mononuclear cells (PBMCs) derived from joint engrafts of the same patients were suspended in serum and engrafted into NOD/SCID mice [4]. Human multilineage hematocytes, including T lymphocytes, B lymphocytes, monocytes, myeloid maturation stages, and primitive progenitor cells, were sustained in xenografted mice for at least 8 weeks. Human rheumatoid factor was detected in the serum of xenografted mice, and invasion of synovium into the implanted cartilage was able to be scored. In this model, the maintenance of an inflammatory microenvironment is successfully achieved as a critical supportive factor for synovial invasion into cartilage.
2 Materials
To obtain explants from RA patients, research protocols should be approved by the Institutional Ethics Committee. All animal experiment protocols should be performed in accordance with the guidelines of the Institutional Animal Care and Use Committee and approved by the committee.
1. Animals: Male NOD/ShiJic-scid (NOD/SCID) mice, 6–10 weeks of age, are used for xenograft experiments (see Note 1).
2. Explants from RA patients: Peripheral blood (20 mL), synovium, bone, and articular cartilage explants can be obtained from RA patients who have undergone prosthetic replacement arthroplasty for therapeutic purposes. All explants should be transferred between institutions or units in a biohazard and cooling container. Explants should be handled for the xenograft procedure as soon as possible after explantation.
10ShuangLiu
3 Methods
3.1 Isolation of PBMC from Peripheral Blood from RA Patients
3. Inhalation anesthesia unit (see Note 2).
4. Centrifuges and centrifuge tubes.
5. Operating table.
6. Warming plate or heating pad.
7. Forceps (fine blunt) and scissors (fine dissection).
8. Syringes, 1 mL.
9. Wound clips and applier.
10. Pipettes and chips.
11. 70% ethanol.
12. Isoflurane or other anesthetics.
13. Histopaque 1077.
14. Cell suspension buffer: Phosphate-buffered saline (PBS), pH 7.2, and 2 mM EDTA. Sterilize the buffer by membrane filtration and keep it cold (2–8°C).
1. For serum collection, collect 2 mL of whole blood into a regular 1.5-mL Eppendorf tube and centrifuge the sample for 15 min at 1500× g at 4 °C. Harvested serum is ready for PBMC suspension.
2. Dilute the remaining whole blood with the same volume of cell suspension buffer.
3. Carefully layer 35 mL of diluted whole blood over 15 mL of Histopaque 1077 in a 50-mL conical tube.
4. Centrifuge at 400× g for 30 min at 20 °C in a swinging bucket rotor without a brake.
5. Harvest the mononuclear cell layer undisturbed at the interphase and carefully transfer the mononuclear cell layer to a new 50-mL conical tube.
6. Fill the conical tube with cell suspension buffer and mix gently. Centrifuge the tube at 300× g for 10 min at 20 °C and carefully remove the supernatant completely.
7. Wash the cells with cell suspension buffer and centrifuge the tube at 300× g for 10 min at 20 °C. Carefully remove the supernatant completely.
8. Resuspend PBMC (1 × 107) using 200 μL of the same patientderived serum for further transplantation.
3.2 Trimming
Explanted Joint Tissue
3.3 Implantation
(See Notes 4 and 5)
1. Keep the explants in saline-wet gauze at 4 °C and use as soon as possible.
2. Trim the explanted synovium and cartilage with bone to a block about 4–6 mm in diameter prior to implantation (see Note 3).
1. Put NOD/SCID mice in an anesthetic induction chamber. Initial induction can be performed using 2.5% isoflurane vaporized in 100% medical oxygen. Following induction, anesthesia should be maintained by placing the mice in front of a small face mask connected to the anesthetic machine using 1% isoflurane vaporized in 100% medical oxygen.
2. Weigh and put mice on the operating table. Place the mouse on its abdomen to expose the back. Shave the back. Use a squirt bottle to apply 70% ethanol to the back and wipe with tissue.
3. Cut the skin with fine dissection scissors, making a 1-cm vertical incision at a point level of the fourth to sixth lumbar vertebrae.
4. After exposing the subcutaneous tissue, the oblique external abdominal muscle is scraped with a scalpel until it bleeds.
5. Put the trimmed RA patient-derived synovium on the oblique external abdominal muscle, and let the connective tissue site of synovium attach to the bleeding muscle.
6. Put the articular cartilage and bone on the synovium (see Note 6) and let the smooth surface of the cartilage touch the articular luminal side of the synovium.
7. Clip the skin together with wound clips or sew up with two or three stiches. Clean the wound with 70% ethanol.
8. Inject serum-suspended PBMC (200 μL), prepared as described in Subheading 3.1, intraperitoneally (see Note 7).
9. At the end of the procedure, put the mouse in a clean cage and place the cage on a warming plate until the mouse recovers from the anesthetic.
3.4
1. Anesthetize the xenografted mice at 6–8 weeks after transplantation (see Note 8).
2. Remove the implanted tissues from xenografted mice, and immerse in 4% paraformaldehyde for tissue fixation. Decalcify and embed the tissues (the protocol can be found in Chap. 5). The sections should be stained with the methods as desired (e.g., hematoxylin and eosin) (Fig. 1).
3. For semi-quantification of synovial invasion into cartilage and bone tissues, sections can be scored from 0 to 4 based on the
Evaluation of Invasion of Synovium
Fig. 1 Histological analysis of implants in xenografted mice. The engrafted tissues were explanted at 8 weeks after transplantation. Following fixation and decalcification, tissue sections were stained using hematoxylin and eosin (original magnification, ×400). Arrows indicate invasion of synovium into implanted cartilage. S synovium, C cartilage
number of invading cell layers and number of invasive sites [4, 5], as follows (see Note 9):
• 0: no or minimal invasion
• 0.5: invasion of 1–2 cells at three independent cartilage sites
• 1: invasion of 3–5 cell layers
• 1.5: invasion of 3–5 layers at three independent cartilage sites
• 2: invasion of 6–10 cell layers
• 2.5: invasion of 6–10 layers at three independent cartilage sites
• 3: invasion of >10 cell layers
• 3.5: invasion of >10 layers at two independent cartilage sites
• 4.5: invasion of >10 layers at three or more independent cartilage sites
4
Notes
1. NOD/SCID mice are characterized by the absence of functional T cells and B cells, deficient natural killer cells, lymphopenia, hypergammaglobulinemia, and a normal hemotopoietic microenvironment. To avoid any unexpected complications, the age of NOD/SCID mice used in the xenograft model should be under 10 weeks. Due to their severely immunocompromised state, NOD/SCID mice should be housed in maximum-barrier facilities. Below are the conditions that we recommend for housing NOD/SCID mice:
• Use microisolator (filter bonneted) or pressurized, individually ventilated cages (PIV/IVC).
• Sterilize or disinfect food, water, bedding, cages, and anything that will come in contact with the mice.
• Only personnel involved in the care of the mice should have access to the mouse room, and caretakers should wear personal protective equipment.
• Before accessing the housing room, operators or caretakers should pass the air shower unit.
• Cages should be changed under a laminar flow hood. Change cages weekly to prevent the introduction of minimum-inoculating doses of opportunistic or commensal organisms into the cage environment.
2. All equipment should be used under sterile conditions.
3. For the evaluation of invasion of synovium, cartilage and bone with normal appearance rather than the lesion site should be chosen.
4. Implantation should be carried out under sterile conditions.
5. NOD/SCID mice could be pretreated by a single intraperitoneal cavity injection of 50 μL anti-asialo-GM1 serum to deplete natural killer cells 1 day before performing xenografting. In our experience, NOD/SCID mice can tolerate the engrafting procedure without any pretreatment.
6. Articular cartilage is always explanted with the bone beneath the cartilage.
7. PBMC can be engrafted by a single injection into the intraperitoneal cavity, intravenous, or intrasplenic injection [6]. The highest amount of human PBMC chimerism can be achieved by intrasplenic injection in NOD/SCID mice. Chimerism of human PBMC is poor using intravenous injection. Considering adverse effects, we chose intraperitoneal injection for engrafting human PBMC.
8. The optimal timing of explantation is strain-and treatmentdependent. A pilot study is required for optimizing the end point of engrafting.
9. Quantification should be carried out on five high-power fields in each section and three sections for each specimen.
Acknowledgment
This work was supported by a Japan Society for the Promotion of Science KAKEMHI Grant (15K19575).
References
1. Rendt KE, Barry TS, Jones DM, Richter CB, McCachren SS, Haynes BF (1993) Engraftment of human synovium into severe combined immune deficient mice. Migration of human peripheral blood T cells to engrafted human synovium and to mouse lymph nodes. J Immunol 151:7324–7336
2. Matsuno H, Yudoh K, Uzuki M, Kimura T (2001) The SCID-HuRAg mouse as a model for rheumatoid arthritis. Mod Rheumatol 11: 6–9
3. Sakuraba K, Fujimura K, Nakashima Y, Okazaki K, Fukushi J, Ohishi M et al (2015) Brief report: successful in vitro culture of rheumatoid arthritis synovial tissue explants at the air-liquid interface. Arthritis Rheum 67:887–892
4. Liu S, Hasegawa H, Takemasa E, Suzuki Y, Oka K, Kiyoi T et al (2017) Efficiency and safety of CRAC inhibitors in human rheumatoid arthritis xenograft models. J Immunol 199: 1584–1595
5. Maeshima K, Yamaoka K, Kubo S, Nakano K, Iwata S, Saito K et al (2012) The JAK inhibitor tofacitinib regulates synovitis through inhibition of interferon-gamma and interleukin-17 production by human CD4+ T cells. Arthritis Rheum 64:1790–1798
6. Zhou W, Ohdan H, Tanaka Y, Hara H, Tokita D, Onoe T et al (2003) NOD/SCID mice engrafted with human peripheral blood lymphocytes can be a model for investigating B cells responding to blood group A carbohydrate determinant. Transpl Immunol 12:9–18
Chapter 3
Scaffolded Chondrogenic Spheroid-Engrafted Model
Shuang Liu
Abstract
Therapeutic approaches using mesenchymal stem cells (MSCs) for a cartilage regeneration strategy are based on their multipotent differentiation for skeletal regeneration. With the utilization of allergenic neutralized type I atelocollagen during the pre-formation of chondrogenic MSC spheroids, cellular condensation and chondrogenic differentiation can be easily achieved. It also benefits the recruitment of host MSCs, which differentiate into chondrocyte-like cells after implantation into the experiment model. Using pre-formed chondrogenic MSC spheroids, the efficacy of anti-rheumatoid agents for cartilage repair can be screened on a large scale ex vivo. Furthermore, atelocollagen-scaffolded chondrogenic spheroids can be utilized for in vivo transplantation into a humanized xenografted arthritis model. Thus, the ability of cartilage self-repair can be qualitatively and quantitatively evaluated.
Because cartilage destruction directly causes joint pain and functional disability, the basic strategy of rheumatoid arthritis (RA) management is to alter the systemic immune status to prevent or delay cartilage destruction. After a prolonged period of time, cartilage and bone damage is almost irreversible, and very limited options remain other than considering prosthetic replacement arthroplasty. Since the regenerative capacity of cartilage is limited due to the slow metabolic rate of chondrocytes, lack of vascularity, and limitation of the number of progenitor cells, how to repair existing damage of cartilage has been challenging [1]. Autologous cellular implantation, including the employment of chondrocytes or multipotency mesenchymal stem cells (MSC), has emerged for joint regeneration [2, 3].
Therapeutic approaches using MSC for a regeneration strategy are based on their immunomodulatory capabilities to achieve systemic immunosuppression and multipotent differentiation for
skeletal regeneration [4]. Human MSC utilized for cartilage regeneration can be obtained from many types of tissues, including bone marrow, synovial tissue, peripheral blood, periosteum, and adipose tissue [5]. Several MSC-based tissue engineering techniques for cartilage regeneration using a pellet culture system or threedimensional (3D) scaffold have been reported [6–8]. Encapsulating cells in a natural biomaterial-based scaffold with a loose framework and high water content, such as collagen, fibrin, hyaluronic acid, or agarose, has been widely studied for 3D spheroid formation [5]. Since collagen molecules are a major component of the car tilage extracellular matrix and are degraded by endogenous collagenases, after removing telopeptide, antigenic-neutralized collagen-based scaffolds provide a suitable background for the application for an articular cartilage repair strategy. Among diverse shapes of scaffolds such as sponge, hydrogel, fibers, and microparticles, collagen-sponge has been utilized for 3D-MSC-derived chondrogenic spheroid formation because of its biocompatibility and capability for maintaining the chondrogenic microenvironment for functional cartilage regeneration [9].
When MSCs are cultured on type I atelocollagen scaffolds, cellular condensation and chondrogenic differentiation are induced. The expression of cartilage-specific markers such as Sox9, type II collagen, and aggrecan was confirmed in atelocollagen-encapsulated MSCs along with a chondrocyte-like appearance [9]. Moreover, type I atelocollagen scaffolds are able to recruit host MSCs in vivo, which can differentiate into chondrocyte-like cells [10, 11]. In this chapter, a laboratory protocol for the establishment of an atelocollagen-scaffolded chondrogenic-MSC-spheroid-based anti-rheumatoid agent screening system is introduced. Using pre-formed chondrogenic MSC spheroids, the efficacy of anti-rheumatoid agents for cartilage repair can be screened on a large scale ex vivo. Furthermore, atelocollagenscaffolded chondrogenic spheroids can be easily utilized for in vivo transplantation into a humanized xenografted arthritis model [12]. Thus, the ability of cartilage self-repair can be qualitatively and quantitatively evaluated.
1. MSC suspension, 2.5 × 105 cells for each spheroid (see Note 1).
3. Human Mesenchymal Stem Cell Functional Identification Kit (#SC006, R&D Systems, Minneapolis, MN), including Chondrogenic Supplement 100× (PART# 390417) and ITS Supplement 100× (PART#390418) (see Note 2).
2.2 Implantation In Vivo
ScaffoldedChondrogenicSpheroid-EngraftedModel19
4. Complete chondrogenic conditioned medium: Poweredby10 Medium containing chondrogenic Supplement 1× and ITS Supplement 1× .
5. AteloCell® Atelocollagen, Honeycomb Disc 96 (Koken, Tokyo, Japan).
6. 2% Atelocollagen Implant (Koken).
7. Round-bottom 96-well cell culture plates (see Note 3).
8. Forceps (fine blunt), sterile conditions.
9. Pipettor and tips, sterile conditions.
10. 37 °C and 5% CO2-incubator suitable for cell culture.
All animal experiment protocols should be reviewed and approved by the Institutional Animal Care and Use Committee.
1. Animals: Male NOD/ShiJic-scid (NOD/SCID) mice, 6–10 weeks of age.
2. MSC-atelocollagen-scaffoldedchondrogenicspheroids (in round-bottom 96-well cell culture plate).
3. Explants from patients used for human xenograft model establishment, including synovium, articular cartilage, and bone explants obtained from patients who underwent prosthetic replacement arthroplasty for therapeutic purposes (see Chap. 2, Subheading 2). Explants should be handled for the xenograft procedure as soon as possible after explantation.
4. Saline-wet gauze at 4 °C.
5. 70% ethanol.
6. Inhalation anesthesia unit.
7. Chondrogenic spheroids.
8. Operating table.
9. Forceps (fine blunt) and scissors (fine dissection).
10. Wound clips and applier.
11. Isoflurane.
12. Phosphate-buffered saline (PBS), pH 7.2.
3 Methods (See Note 4)
3.1 3D Culture of Chondrogenic Spheroids
1. Using fine pointed curved forceps, carefully set Atelocollagen Honeycomb Discs onto a round-bottom 96-well cell culture plate.
2. Immerse the Honeycomb sponge in 50 μL of 2% Atelocollagen Implant (see Note 5).
3. Incubate the matrix at 37 °C for 1 h.
3.2 Implantation In Vivo (See Note 7)
Fig. 1 Evaluation of chondrogenic micromass in vitro. Atelocollagen-scaffolded MSC spheroids were pre-formed by 21 days of culture in a chondrogenic conditioned medium containing methotrexate (MTX) at doses of 0, 0.01, 0.1, and 1 μM. Scanning of micromass was performed using an MR imaging and analytic system for small animals. T2-weighted scout images were acquired and reconstructed as a three-dimensional image. The volume of the region of interest could be easily achieved
4. Add 200 μL of prewarmed Poweredby10 Medium into the well.
5. Calibrate the formed matrix at 37 °C in a humidified atmosphere with 5% CO2 overnight.
7. Remove the Poweredby10 Medium in the well and carefully seed MSC on the pre-formed matrix.
8. Culture the atelocollagen-scaffolded spheroids for 21 days. Change the culture medium with freshly prepared complete chondrogenic differentiation medium every 3 days (see Note 6).
9. By directly applying drugs into the chondrogenic conditioned medium and quantifying the volume of 3D-formed micromass, atelocollagen-scaffolded chondrogenic spheroids could be directly utilized for evaluation of efficacy ex vivo (Fig. 1).
1. Trim the synovium and cartilage with bone to a block about 4–6 mm in diameter prior to implantation. Keep the explants in saline-wet gauze at 4 °C.
2. Put NOD/SCID mice in an anesthetic induction chamber. Initial induction can be performed using 2.5% isoflurane vaporized in 100% medical oxygen. Following induction, anesthesia should be maintained by placing the mice in front of a small face mask connected to an anesthetic machine using 1% isoflurane vaporized in 100% medical oxygen.
3. Put mice on the operating table. Place the mouse on its abdomen to expose the back. Shave the back. Use a squirt bottle to apply 70% ethanol to the back and wipe with tissue.
4. Cut the skin with fine dissection scissors, making a 1.5 cm longitudinal incision at the level of the fourth to sixth lumbar vertebrae.
5. After exposing the subcutaneous tissue, the oblique external abdominal muscle is scraped with a scalpel until it bleeds.
6. Put the trimmed synovium on the oblique external abdominal muscle, and let the connective tissue site of synovium attach to the bleeding muscle.
7. Briefly rinse a chondrogenic spheroid in PBS and carefully set the spheroid on the synovium (Fig. 2a).
Fig. 2 Implantation and evaluation of chondrogenic spheroid in a xenograft model. (a) Implants for xenograft model. Two pieces of in vitro pre-formed atelocollagen-scaffolded chondrogenic MSC spheroid are set on patient-derived synovium. The explanted articular cartilage and bone should be set on the spheroid, with the smooth surface of the cartilage touching the spheroid on the articular luminal side. A representative image of hematoxylin and eosin staining of xenografted-implants (b) without chondrogenic MSC spheroid and (c) with chondrogenic MSC spheroid 8 weeks after implantation is shown. Invasion of synovium into cartilage was observed in the xenograft model without chondrogenic spheroid implantation, while the cartilage damage was repaired and cartilage with a smooth surface was maintained in the chondrogenic MSCspheroid-implanted xenograft model. C patient-derived cartilage, CS chondrogenic MSC spheroid, S patient-derived synovium. Scale bar: 100 μm
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Merry’s Adventures.
.
I easily make my readers, who have always lived in cities or towns, understand the pleasure of sleeping in the woods, with no roof but the sky. Perhaps most persons would think this a hardship, and so it would be, if we had to do it always: but by way of adventure now and then, and particularly when one is about seventeen, with such a clever fellow as Mat Olmsted for a companion and a guide, the thing is quite delightful.
The affair with the panther had excited my fancy, and filled my bosom with a deep sense of my own importance. It seemed to me that the famous exploits of Hercules, in Greece, which are told by the old poets, were, after all, such things as I could myself achieve, if the opportunity only should offer
Occupied with these thoughts, I assisted Mat in collecting some fagots for our night fire—but every moment kept looking around, expecting to see some wild animal peeping his face between the trunks of the gray old oaks. In one instance I mistook a stump for a bear’s head, and in another I thought a bush at a little distance, was some huge monster, crouching as if to spring upon us.
The night stole on apace, and soon we were surrounded with darkness, which was rendered deeper by the fire we had kindled. The scene was now, even more wild than before: the trees that stood around, had the aspect of giants, lifting their arms to the sky;—and their limbs often assumed the appearance of serpents, or demons, goggling at us from the midnight darkness. Around us was a seeming tent, curtained with blackness, through which not a ray of light could penetrate.
I amused myself for a long time, in looking at these objects, and I remarked that they assumed different aspects at different times—a thing which taught me a useful lesson, and which I will give, gratis, to my young readers. It is this, that fancy, when indulged, has the power to change objects to suit its own wayward humor. Whoever wishes to be guided right, ought, therefore, to beware how he takes fancy for a guide.
When our fire had been burning for about half an hour, Matthew having unbuckled his pack, took out some dried deer’s flesh, upon which we made a hearty supper: we then began to talk about one thing and another, and, finally, I spoke of the Indians, expressing my curiosity to know more about them. Upon this, Mat said he would tell an Indian story, and accordingly, he proceeded nearly as follows:
These six nations, you must know, were not originally confined to this small tract of country, but they were spread far and wide over the land. Nor were they always united, but in former days they waged fierce wars with one another. It was the custom among all the tribes to put captives to death, by burning them, inflicting at the same time the most fearful tortures upon the victims. Sometimes, however, they adopted the captive, if he showed extraordinary fortitude, into the tribe, and gave him all the privileges of the brotherhood.
An instance of this sort occurred with the Senecas. They had been at war with the Chippewas, who lived to the north. Two small bands of these rival tribes met, and every one of the Chippewas was slain, save only a young chief named Hourka. He was taken, and carried to the village of the victorious Senecas. Expecting nothing but torture and death, he awaited his fate, without a question, or a murmur. In a day or two, he saw the preparations making for his sacrifice: a circular heap of dried fagots was erected, and near it a stake was driven in the ground.
To this he was tied, and the fagots were set on fire. The scorching blaze soon flashed near his limbs, but he shrunk not. An Indian then took a sharp piece of stone, and cut a gash in Hourka’s side, and inserted in it a blazing knot of pine. This burned down to the flesh, but still the sufferer showed no signs of distress. The people of the
tribe, came around him, and jeered at him, calling him coward, and every other offensive name: but they extorted not from him an impatient word. The boys and the women seemed to be foremost in taunting him; they caught up blazing pieces of the fagots, and thrust them against his naked flesh; but yet, he stood unmoved, and his face was serene, showing, however, a slight look of disdain. There was something in his air which seemed to say, “I despise all your arts—I am an Indian chief, and beyond your power.”
Now it chanced that a daughter of an old chief of the Senecas, was there, and her heart was touched with the courage and manly beauty of the youthful Chippewa; so she determined to save his life if she could: and knowing that a crazy person is thought by the Indians to be inspired, she immediately pretended to be insane. She took a large fragment of the burning fagot in her hand, and circling around Hourka, screamed in the most fearful manner. She ran among the woman and boys, scattering the fire on all sides, and at the same time exclaiming, “Set the captive free,—it is the will of Manitto, the Great Spirit!”
This manoeuvre of the Indian maiden was so sudden, and her manner was so striking, that the Indians around were taken by a momentary impulse, and rushing to the captive, sundered the strings of bark that tied him to the stake, and, having set him at liberty, greeted him as a brother. From this time, Hourka became a member of the tribe into which he was thus adopted, and none treated him otherwise than as a chief, in whose veins the blood of the Senecas was flowing, save only a huge chief, called Abomico.
This Indian was of gigantic size, and proportionate power. He had taken more scalps in fight, than any other young chief, and was, therefore, the proudest of all the Senecas. He was looked upon by the girls of the tribe, very much as a young man is among us, who is worth a hundred thousand dollars. When, therefore, he said to Meena—the daughter of the chief who saved the life of Hourka—that he wanted her for his wife, he was greatly amazed to find that she did not fancy him. He went away wondering that he could be refused, but determining to try again. Now the long, dangling soaplocks, and filthy patches of beard, worn by our modern dandies,
who desire to dazzle the eyes of silly girls—were not in vogue among the Senecas: but foppery is a thing known among savages as well as civilized people.
Accordingly, Abomico, when he had determined to push his suit with Meena, covered himself entirely over with a thick coat of bear’s grease; he then painted one side of his face yellow, the other blue; his arms he painted red; on his breast he drew the figure of a snake; on one leg he painted a skunk; on the other a bear. Around his neck he hung a necklace of bears’ claws, and on his arm he bore forty bloody scalps, which he had taken from the heads of enemies slain in battle; at his back was a quiver of arrows, and in his left hand was a bow. In his hair was stuck a bunch of eagles’ feathers; from his right ear swung the skin of a racoon; in his right hand he bore the wing of a crow.
Thus attired, Abomico marched toward the tent, where Meena dwelt with her father. Never was a beau of one of our cities, new from the hands of the tailor, more delighted with his appearance, than was this Indian dandy, as he drew near to the tent, and waited at the door for the maiden to appear “If she can resist my charms now,”—thought Abomico,—“she must be bewitched indeed!”
Meena soon appeared—and the chief spoke to her again, begging her to become his wife. “Come!” said he—“go with me, and be the singing bird in my nest. I am a great warrior. I have slain forty brave men in battle. I have feasted on the flesh, and drunk the warm blood, of my enemies. I have the strongest arm, the truest hand, the swiftest foot, the keenest eye, of any chief in the mighty tribe of the Senecas.”
“It is not true!” said Meena.
“Not true?” said the chief, in great anger and astonishment. “Who dares to match himself with Abomico? Who can vie with him in the race? Who can shoot with him at the mark? Who can leap with him at the bar?”
“Hourka!” said Meena.
“It is a lie,” said Abomico; though I must say, that he meant no offence—because, among the Indians, such a speech was not a discourtesy.
“Nay—nay,” said Meena—“I speak the truth; you have come to ask me to be your wife. Hourka has made the same request. You shall both try your power in the race and the leap, and at the bow. He who shall be the master in the trial, may claim Meena for his slave.”
This proposition was gladly accepted, and Hourka being informed of it, a time for the trial was appointed. The people of the village soon heard what was going on; and, as the Indians are always fond of shows and holidays, they rejoiced to hear of the promised sport.
The day of the trial arrived. In a grassy lawn, the sport was to be held; and here the throng assembled. It was decreed by the chiefs that the first trial should be with the bow. A large leaf was spread out upon a forked branch of a tree, and this was set in the ground, at the distance of about fifty yards. Abomico shot first, and his arrow pierced the leaf, within half an inch of the centre. Hourka followed, and his arrow flew wide from the mark, not even touching the leaf. He seemed indeed careless, and reckless. But, as he turned his eye upon Meena, he saw a shade of sorrow come over her face.
In an instant the manner of the young chief changed. He said to himself,—“I have been mistaken: I thought the maiden slighted me and preferred my rival: but now I know that she loves me, and I can now beat Abomico.”
There were to be three trials of the bow In the two which followed the first, which we have described, Hourka had the advantage and was pronounced the victor. And now came the leap. A pole was set horizontally upon stakes, to the height of about five feet, and Hourka, running a little distance, cleared it easily. Abomico followed, and he also leaped over it with facility. It was then raised about a foot, and Hourka, bounding like a deer of the wood, sprang over the pole, amid the admiring shouts of the multitude. Abomico made a great effort, and he too went over, but his foot grazed the piece of wood, and the victory here again was awarded to Hourka.
The face of the haughty Abomico, now grew dark as the thundercloud. He could bear to be rejected by Meena; but to be thus vanquished before the whole tribe, and that too by one who had not the real blood of a Seneca, was more than his pride could bear. He was, therefore, plotting some scheme of revenge, when the race was marked out by the chiefs. It was decreed that they should run side by side to a broad river which was near; that they should swim across; ascend on the opposite bank to a place above a lofty cataract in the river, and recrossing the river there, return to the point of their departure.
The place occupied by the spectators, was so elevated as to command a fine view of the entire race-ground; and the interest was intense, as the two chiefs departed, bounding along, side by side, like two coursers. The race was long nearly equal. They came to the river, and at the same moment both plunged into the water. They swam across, and at the same moment clambered up the rocky bank on the other shore. Side by side they ran, straining every muscle. They ascended to the spot above the roaring cataract, and plunged into the river; then drew near the place where the water broke over the rocks in a mighty sheet, making the earth tremble with the shock of their fall. Still the brave swimmers heeded not the swift current that drew them toward the precipice. Onward they pressed, cutting the element like ducks, and still side by side.
Intense was the interest of the spectators, as they witnessed the strife. But what was their amazement, when they saw Abomico rise above the wave, grapple Hourka and drag him directly toward the edge of the cataract. There was a shout of horror, through the tribe, and then a deathlike silence. The struggle of the two rivals was fearful, but in a short space, clinging to each other, they rolled over the precipice, and disappeared among the mass of foam, far and deep below!
Killed, by falling on the rocks, and gashed by many a ghastly wound, the huge form of Abomico was soon seen drifting down the stream; while Hourka swam to the shore, and claimed his willing bride, amid the applauses of men, women and children.
The Zodiac.
T Zodiac consists of a broad belt in the heavens, among which the sun appears to make his annual circuit. The stars are arranged in groups, and the ancients, who were fond of astronomy, called these groups or constellations, by particular names. One group they called ursa major, or great bear; one they called orion; another, the crown; another, the dog; another Hercules, &c.
In the month of March, the sun is said to enter aries, that is the group or constellation called aries, or the ram; in April it enters taurus, or the bull; in May, gemini, the twins; in June, cancer, the crab; in July, leo, the lion; in August, virgo, the virgin; in September, libra, the scales; in October, scorpio, the scorpion; in November,
sagittarius, the archer; in December, capricorn, the goat; in January, aquarius, the water bearer; in February, pisces, the fishes.
The Voyages, Travels, and Experiences of Thomas Trotter.
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The grotto of Pausilippo.—A dying man.—The Lazzaroni.—Weather at Naples.—The grotta del cane.—Inhuman sport.—Subterranean fires.—A Funeral.—Characteristics of the Neapolitans.
I heard a great deal of the grotto of Pausilippo, which is a great tunnel through a mountain at one end of the city, and I took a walk toward that quarter, for the purpose of visiting it.
This is certainly one of the most surprising works of art in the world, considering its age. It was executed two or three thousand years ago, and is probably the most permanent artificial work on the face of the earth. Even the Egyptian pyramids will not last so long as this. To have some idea of it, you must understand that Naples is separated from the towns on the northern coast by the hill of Pausilippo, which is a ridge of solid rock.
Through this rock an immense tunnel is cut, three quarters of a mile long, and nearly a hundred feet high. It is broad enough for two carriages to pass, and lighted by lamps. Several air-holes, at proper distances, serve to ventilate it and keep the air pure. A great deal of travel is constantly passing through it: and during the heat of summer, the grotto, has a most refreshing coolness. The rumbling of the carriages is echoed from the rocky vault overhead in a very remarkable manner. Altogether, the place struck me with surprise and astonishment; and when I thought of our railroad tunnels, which we boast of as modern inventions, I could not help repeating the observation of king Solomon, that “there is no new thing under the sun.”
While I sat at supper in the evening, I was startled by hearing a bell tinkling violently under my window. I ran to the balcony and found the whole street in a blaze of light. A religious procession was going down the street bearing lighted tapers. I was told that it was a priest going to administer extreme unction to a dying man.
At the sound of the bell, which was carried by one of the procession, all the neighbors ran to the windows and balconies with lamps and candles, and fell upon their knees; for this is the custom on such occasions. In an instant the whole street was in a blaze of light, and the prospect of this illumination, with the long procession of persons dressed in white, chanting a mournful dirge, and the crowds in the balconies in solemn and devout attitudes, struck me very forcibly. As the procession passed by each house, the spectators crossed themselves and uttered a prayer for the soul of the dying man. So sudden are the transitions of these people from the gayety and merriment of their daily occupations to the solemnity of their religious observances.
Everybody who has been at Naples, has something to say about the Lazzaroni, which is the name given to the idle fellows and ragamuffins of this city. Many people imagine them to be a distinct race of men, like the gipseys in other parts of Europe; but this is an error. Every city in Europe has its proportion of lazy and ragged fellows: but in Naples their number is so great that they have obtained this peculiar name. By some, their numbers are stated at twenty thousand. I will not vouch for the full number, but they exist in swarms. Nowhere else did I ever see such comical raggedness as among these people. The scarecrows, which Yankee farmers set in their cornfields to frighten away the birds, are genteel figures compared to these fellows. One has half a pair of trowsers; another half a jacket, and no trowsers at all; another wears the leg of an old stocking for a cap; another has a ragged pair of breeches the wrong side upwards for a shirt. As to the patches and tatters, they surpass all power of language to describe. How they get their living, one is puzzled to guess, for they seem to spend all the day basking in the sun; and in spite of their rags and dirt, they appear to be as happy as lords. They are constantly in good humor, singing, chattering,
grimacing, and cutting capers from morning to night. In fact, notwithstanding their want of almost all those things which we call necessaries of life, they appear to be troubled with very little suffering. Their rags and nakedness give them little concern, for the climate is so mild that they hardly feel the want of a covering. Their food is chiefly macaroni, which is very cheap here: two or three cents worth will suffice a man for a day. Their manner of eating it makes a stranger laugh; they hold it up in long strings, at arm’s length, and swallow it by the yard at a time. As for their homes, the most of them have none: they sleep in the open air, on the steps of the churches, and wherever they can find a convenient spot to lie.
It was about the middle of March, which is the most disagreeable month of the whole year in this country; yet I found the weather very mild and pleasant. Light showers of rain happened almost every day; but these lasted commonly but a few minutes and were succeeded by warm sun-shine. I could discern the Appenines at a distance, covered with snow, while the hills around the city were decked with green olive trees. Oranges and lemons were plenty and very cheap: three or four for a cent.
I set out on a walk to visit the famous grotta del cane, or “dog’s cavern,” which is only a few miles from Naples. The road lay through the grotto of Pausilippo, and I could not avoid again admiring this wonderful cavern, the work of men who lived in what we have supposed to be an age of barbarism. At the further end I emerged into the open air and found a region of fields and vineyards, separated by walls of clay. Little children ran along by my side, tumbling head over heels, clacking their chops, making queer noises and antic gestures by way of begging for coppers. All along the road were poplar trees, to which the vines were trained, but they were not in leaf. After a walk of three or four miles I came to lake Agnaro, a piece of water about the size of Fresh Pond in Cambridge. On the shore of this lake is the grotta del cane. It is a rocky cavern which enters horizontally a little above the water, and emits from its mouth a sulphureous steam or vapor, which will kill a dog if he is put into the cavern. People who live in the neighborhood keep dogs for the purpose of exhibiting this phenomenon to strangers. The dogs know
the fatal properties of this cave, and refuse to go in. While I was there, some of these fellows came to me and offered to exhibit the experiment; but I declined, not wishing to see an animal treated with cruelty for mere curiosity. They assured me that the dog need not be killed—that they would only keep him in the cave long enough to throw him into a swoon, and then bring him to life again by plunging him into the water. I told them this was as bad as killing him outright: for the animal could suffer no more by actually dying. They were very unwilling to lose their expected fee, and answered me that there was no suffering in the case, but, on the contrary, the dogs were very fond of the sport! I laughed at this impudent falsehood, and refused to have anything to do with the exhibition.
A few minutes after, a party of visiters arrived who had no such humane scruples: they were resolved to see the experiment tried. Accordingly, a dog was brought forward; and I now had a chance to see how much truth there was in the assertion that these animals were fond of being choked to death. The poor dog no sooner perceived his visiters than he became as perfectly aware of what was going forward as if he had heard and understood every syllable that had been said. It showed the utmost unwillingness to proceed towards the cavern, but his master seized him by the neck and dragged him with main force along till he reached the mouth of the cave, into which he thrust him howling and making the most piteous cries. In a few minutes he fell upon the ground motionless, and lay without any signs of life. The spectators declared that they had seen enough to satisfy them; on which the fellow took the dog up by the ears and plunged him into the lake. After two or three dips, the poor animal began to agitate his limbs and at length came to himself and ran scampering off. These inhuman exhibitions ought not to be encouraged by travellers.
Every part of the neighborhood of the city abounds with evidence of the existence of volcanic fire, under ground. As I walked along the road I found the smoke issuing from holes and clefts in the ground: and on placing my hands in these fissures, I found them so hot that one might roast eggs in them. Yet people build houses and pass their lives upon these spots, without troubling themselves with the
reflection that they live on a thin crust of soil hanging over a yawning gulf of fire! In my walk homeward I passed by a hill, about the size of Bunker Hill, which some time ago rose up suddenly, in a single night, from a level plain. It is now all overgrown with weeds and bushes. If it were not for Mount Vesuvius, which affords a breathing-place for these subterranean fires, it is highly probable that the whole face of the country would be rent into fragments by earthquakes and volcanic explosions. Vesuvius may be called the safety valve of the country.
On my way home, I was stopped on the road by an immense crowd. It was a funeral. A long train of monks and priests attended the hearse, each one clad in a dress which resembled a loose white sheet thrown over the head and falling down to the feet, with little round holes cut for the eyes. They looked like a congregation of spectres from the other world. The corpse was that of an army officer. He lay not in a coffin, but exposed in full uniform upon a crimson pall edged with gold. Everything accompanying the hearse was pompous, showy and dazzling.
This indeed is the characteristic of the people; almost everything in their manners and mode of life is calculated to strike the senses and produce effect by dazzling and external display. Nothing can surpass the splendor of their religious processions, the rich and imposing decoration of their churches, and the pomp and parade and showy display which attend the solemnization of all their public festivals. The population of these countries are exceedingly sensitive to the effect of all these exhibitions, and their lively and acute feelings bring them under the influence of whatever is addressed strongly to their outward senses. They are little guided by sound reason and sober reflection, but are at the mercy of all the impulses that arise from a keen sensibility and an excitable imagination.
Story of Philip Brusque.
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The meeting.—Discussion.—A government adopted.—Conclusion for the present.
T time for the meeting of the people to take measures for the establishment of a government for the island of Fredonia, was fixed for the day which followed the events narrated in the last chapter. This meeting was looked forward to with intense interest, by all parties. The men, who knew that there could be no peace or safety in society, without government, regarded the event as likely to decide whether the inhabitants of the island were to be happy or miserable.
The women, who were perhaps not apt to reflect upon these things, had also learned from their experience that a government, establishing and enforcing laws, was indispensable to the quiet and security of society: they saw that their own lives, their freedom, their homes, were not secure, without the protection of law. Even the children had found that government was necessary, and these as well as the women, were now rejoicing at the prospect of having this great blessing bestowed upon the little community of Fredonia.
The day for the meeting arrived, and the men of the island assembled, agreeably to the appointment. First came the men of the tent party, and then, those from the Outcast’s cave. The latter were greeted by a shout of welcome, and mingling with the rest, a kind shaking of hands took place between those, who so lately were arrayed against each other in deadly conflict.
After a short time, Mr. Bonfils, being the oldest man of the company, called the assembly to order, and he being chosen
chairman, went on to state the objects of the assembly, in the following words:
“My dear friends; it has been the will of Providence to cast us together upon this lonely, but beautiful island. It would seem that so small a community, regulated by mutual respect and mutual good will, might dwell together in peace and amity, without the restraints of law, or the requisitions of government. But history has told us, that in all lands, and in all ages, peace, order, justice, are only to be secured by established laws, and the means of carrying them into effect. There must be government, even in a family; there must be some power to check error, to punish crime, to command obedience to the rule of right. Where there is no government, there the violent, the unjust, the selfish, have sway, and become tyrants over the rest of the community. Our own unhappy experience teaches us this.
“Now we have met together, with a knowledge, a conviction of these truths. We know, we feel, we see that law is necessary, and that there must be a government to enforce it. Without this, there is no peace, no security, no quiet fireside, no happy home, no pleasant society. Without this, all is fear, anxiety, and anarchy
“Let us then enter upon the duties of this occasion, with a proper sense of the obligation that rests upon us; of the serious duty which is imposed on every man present. We are about to decide questions which are of vital interest, not only to each actor in this scene, but to these wives and sisters and children, whom we see gathered at a little distance, watching our proceedings, as if their very lives were at stake.”
This speech was followed by a burst of applause; but soon a man by the name of Maurice arose—one who had been a leading supporter of Rogere—and addressed the assembly as follows:
“Mr. Chairman; it is well known that I am one of the persons who have followed the opinions of that leader who lost his life in the battle of the tents. I followed him from a conviction that his views were right. The fact is, that I have seen so much selfishness in the officers of the law, that I have learned to despise the law itself. Perhaps, however, I have been wrong. I wish to ask two questions—the first is
this: Is not liberty a good thing? You will answer that it is. It is admitted, all the world over, that liberty is one of the greatest enjoyments of life. My second question then is—Why restrain liberty by laws? Every law is a cord put around the limbs of liberty. If you pass a law that I shall not steal, it is restraint of my freedom; it limits my liberty; it takes away a part of that, which all agree is one of the greatest benefits of life. And thus, as you proceed to pass one law after another, do you not at last bind every member of society by such a multiplied web of restraints, as to make him the slave of law? And is not a member of a society where you have a system of laws, like a fly in the hands of the spider, wound round and round by a bondage that he cannot burst, and which only renders him a slave of that power which has thus entangled him?”
When Maurice had done, Brusque arose, and spoke as follows:
“Mr. Chairman; I am happy that Mr. Maurice has thus stated a difficulty which has arisen in my own mind: he has stated it fairly, and it ought to be fairly answered. Liberty is certainly a good thing; without it, man cannot enjoy the highest happiness of which he is capable. All useless restraints of liberty are therefore wrong; all unnecessary restraints of liberty are wrong. But the true state of the case is this: we can enjoy no liberty, but by submitting to certain restraints. It is true that every law is an abridgment of liberty; but it is better to have some abridgment of it, than to lose it all.
“I wish to possess my life in safety; accordingly I submit to a law which forbids murder: I wish to possess my property in security; and therefore I submit to a law which forbids theft and violence: I wish to possess my house without intrusion; I therefore submit to a law which forbids one man to trespass upon the premises of another: I wish to go and come, without hindrance, and without fear; I therefore submit to a law which forbids highway robbery, and all interference with a man’s pursuit of his lawful business.
“Now, if we reflect a little, we shall readily see that by submitting to certain restraints, we do actually increase the amount of practical, available, useful liberty. By submitting to laws, therefore, we get more freedom than we lose. That this is the fact, may be easily
tested by observation. Go to any civilized country, where there is a settled government and a complete system of laws, and you will find, in general, that a man enjoys his house, his home, his lands, his time, his thoughts, his property, without fear: whereas, if you go to a savage land, where there is no government and no law, there you will find your life, property, and liberty, exposed every moment to destruction. Who, then, can fail to see that the very laws which abridge liberty in some respects, actually increase the amount of liberty enjoyed by the community.”
Maurice professed himself satisfied with this solution of his difficulties; and the meeting proceeded to appoint a committee, to go out and prepare some plan, to be submitted to the meeting. This committee returned, and after a short space, brought in a resolution, that Mr. Bonfils be for one year placed at the head of the little community, with absolute power; and that, at the end of that period, such plan of government as the people might decree, should be established.
This resolution was adopted unanimously. The men threw up their hats in joy, and the air rang with acclamations. The women and children heard the cheerful sounds, and ran toward the men, who met them half way. It was a scene of unmixed joy. Brusque and Emilie met, and the tears of satisfaction fell down their cheeks. François went to his aged mother, and even her dimmed eye was lighted with pleasure at the joyful issue of the meeting.
We must now take leave of the island of Fredonia—at least for a time—and whether we ever return to it, must depend upon the wishes of our young readers. If they are anxious to see how the people flourished under the reign of their aged old chief, and how they proceeded in after years, perchance we may lift the curtain and show them the scene that lies behind it. But I hope that our readers have learnt, that not only men and women, but children, have an interest in government, and therefore that it is a thing they should try to understand.
The Tanrec.
T creature resembles the hedgehog, but is larger than that animal, and is destitute of a tail. It does not roll itself into a ball, for defence, like the former animal. It passes three of the warmest months of the year in a state of torpor, differing in this respect from other animals, which become torpid from extreme cold. Its legs are very short, and it moves very slowly. It is fond of the water, and loves to wallow in the mud. It moves about only by night. There are three species, all found in the island of Madagascar.
Letter from a Correspondent.
Little Readers of the Museum:
I sometimes read Mr Robert Merry’s Museum, and I like it very much, as I presume all his little “blue-eyed and blackeyed readers” do. He talks very much like good old Peter Parley. I should think he had heard him tell many a story while he rested his wooden leg on a chair, with a parcel of little laughing girls and boys around him. Oh, how many times I have longed to see him, and crawl up in his lap and hear his stories! But Mr. Merry says he is dead, and I never can see him. I am very—very sorry, for I hoped I should sometime visit him, for I loved him very much, and I guess he would have
loved me some, for I like old people, and always mean to treat them with respect. How cruel it was for others to write books and pretend that Peter Parley wrote them!—for it seems that this shortened his life. I am glad, however, that Mr. Merry has his writings, for I think he loves his little friends so well that he will frequently publish some of them. I said that I loved Peter Parley, and I guess you will not think it strange that I should, when I tell you what a useful little book he once published, and how much pleasure I took in reading it. He wrote a great many interesting pieces which I read and studied, and they did me much good, I think. I hope that the little readers of the Museum will learn a good deal from what they read.
Peter Parley wrote a piece which told us how to make pens. I read it over, and over again, and, finally, I thought I would see if I could not make one. So I went to my little desk and took out a quill, got my aunt’s knife and laid the book before me and tried to do just as Peter Parley told me I must. I succeeded very well, and my friends were quite pleased. This encouraged me very much, and soon I made them so well that my teachers made me no more pens. By-and-by my little associates got me to make and mend theirs, and I loved the business very much.
Well, a few years since, I went to a beautiful village to attend school, where a splendid academy stands, around which, are large green trees, under whose shade my little readers would love to sit. There I staid two or three years. Often did I walk out with the teachers, whom I loved, to botanize, or ramble, with nimble step, over the beautiful hills of that sweet place, and listen to the constant murmur of its waterfalls, or gather the delicate flowers that grew so plentifully there. But to my story. My teachers saw that I made my own pens, and occasionally, when they were busy, would bring me one to make for them. The students soon found it out, and I had plenty of business. One day the principal of the school came to me and offered to compensate me by giving me my tuition one term, which was six dollars, if I would make
and mend pens. I did not accept the money of course, though I cheerfully and gladly performed the small service.
So you see, Peter Parley’s instruction has done me a great deal of good, for how many persons there are who cannot make a good pen, because they never learned how.
My little readers, I am now almost twenty years old, but I still remember many other things which I read in Peter Parley’s books when I was a little girl. Mr. Robert Merry talks and writes just like him, almost, and I hope you will love to read and study attentively Merry’s Museum, for it is a good little work, and a pleasant one. Be assured, my young friends, you can learn a great deal from it, if you read it carefully. I should like to say much more to you, but I cannot now. I have been sitting by the fire, in a rocking-chair, writing this on a large book, with a pussy under it for a desk, but she has just jumped from my lap, and refuses to be made a table of any longer. So farewell.
Your young friend, L .
Springfield, Jan. 6, 1842
C B .—“Has that cookery book any pictures?” said Miss C. to a bookseller. “No, miss, none,” was the answer. “Why,” exclaimed the witty young lady, “what is the use of telling us how to make a good dinner, if they give us no plates?”
