The Rabbit as a Model for Studying Lung Disease and Stem Cell

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 691830 12 pageshttpdxdoiorg1011552013691830

Review ArticleThe Rabbit as a Model for Studying Lung Disease andStem Cell Therapy

Nurfatin Asyikhin Kamaruzaman Egi Kardia Nurulain lsquoAtikah KamaldinAhmad Zaeri Latahir and Badrul Hisham Yahaya

Cluster for Regenerative Medicine Advanced Medical amp Dental Institute (AMDI) Universiti Sains Malaysia Bandar Putra Bertam13200 Kepala Batas Malaysia

Correspondence should be addressed to Badrul Hisham Yahaya badrulamdiusmedumy

Received 17 December 2012 Revised 28 February 2013 Accepted 28 February 2013

Academic Editor Thomas Liehr

Copyright copy 2013 Nurfatin Asyikhin Kamaruzaman et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

No single animal model can reproduce all of the human features of both acute and chronic lung diseases However the rabbitis a reliable model and clinically relevant facsimile of human disease The similarities between rabbits and humans in terms ofairway anatomy and responses to inflammatory mediators highlight the value of this species in the investigation of lung diseasepathophysiology and in the development of therapeutic agents The inflammatory responses shown by the rabbit model especiallyin the case of asthma are comparable with those that occur in humansThe allergic rabbit model has been used extensively in drugscreening tests and this model and humans appear to be sensitive to similar drugs In addition recent studies have shown that therabbit serves as a good platform for cell delivery for the purpose of stem-cell-based therapy

1 Introduction

Public awareness of lung disease has grown tremendously inthe last several decades People can experience either acuteor chronic disease Acute lung disease describes conditionsof abnormal lung function when exposed to certain stimuliand it can have severe effects on human health Acute lunginjury (ALI) and acute respiratory distress syndrome (ARDS)are clinical syndromes that are defined by varying degrees ofventilation perfusionmismatch acute hypoxemic respiratoryfailure bilateral pulmonary infiltrates (oedema and normalcardiac filling pressure) and poor lung compliance [1 2]Thisfailure can be classified as life-threatening respiratory failuredue to lung injury caused by a variety of precipitants

The development of ALIARDS can be either direct orindirect with development patterns differing in terms ofthe route of the injury to the lung (Figure 1) Pulmonaryoedema refers to the influx of protein-rich fluid into thealveolar spaces resulting from the increased permeability ofthe alveolar-capillary barrier [3] and it is often observedin the early stage of ALIARDS Oedema is categorised asa direct factor when the injurious agent reaches the lung

through the airways or by trauma to the chest and as anindirect factor when the injurious agent reaches the lungthrough the bloodstream [4]

In the early stage of ALIARDS development the pathol-ogy of the patientrsquos lung is accompanied by increased capillarypermeability alveolar and pulmonary oedema and necrosisof the alveoli [4 5] Common clinical features in patientswith pulmonary oedema are shortness of breath coughingup blood or bloody froth difficulty breathing when lyingdown a feeling of ldquoair hungerrdquo or drowning and grunting orwheezing sounds with breathing

ALI which is the mildest form of ARDS develops in theearly stages of lung disease Any stimulus that can initiatesystemic or local inflammation can trigger the onset ofALI in a person ARDS is characterised by the diffusion ofalveolar damage alveolar capillary leakage and protein-richpulmonary oedema all of which lead to clinical observa-tions of poor lung function severe hypoxemia and bilateralinfiltrates on chest radiographs [2] ARDS often develops inpatients diagnosed with sepsis pneumonia and trauma thathas been treated with multiple transfusions Patients diag-nosed with ARDS are usually mechanically ventilated when

2 BioMed Research International

Direct factors

Indirect factors

Infiltration recruitment of

inflammatory cells and formation of

granulation tissue (extracellular matrix

fibroblasts endothelial cells neutrophils and

leukocytes)

Acute lung injury acute respiratory distress syndrome

Lung injury

∙ Lung infection∙ Aspiration of

stomach contentinto lung∙ Inhalation of

highly toxic gases

∙ Sepsis∙Multiple trauma

with shock∙ Large volume

blood replacement

Figure 1 Direct and indirect factors that contribute to the onset of acute lung disease especially acute lung injury and acute respiratorydistress syndrome

the attacks occur Common clinical conditions associatedwith the onset of ARDS are pneumonia aspiration of gastriccontents pulmonary contusion fat emboli near drowninginhalation injury and reperfusion pulmonary oedema aftertransplantation or pulmonary embolectomy [5] Workers inchemical producing factories can also develop ALIARDSdue to inhalation of noxious fumes

Other than harsh chemical agents and biological factorphysical force on airway lumen also can induce hyperrespon-siveness In a study done by Latahir and Yahaya [6] they useda novel brushing technique via tracheal perturbation usingan endotracheal tube to induce injury on the upper airwayThis technique applied amount of forces on the wall of thetrachea which caused bleeding in the area of injury Later onduring the recovery period the recruitment of neutrophilsinto the area of injury will lead to the onset of acute lunginjury This novel technique can be used as an alternativemethod to induce acute lung injury on airway lumen withoutdoing minor surgery which requires skill

Asthma chronic obstructive lung disease (COPD)chronic bronchitis emphysema bronchiectasis and cysticfibrosis are the primary examples of the pathological con-sequences of chronic lung disease Smoke inhalation is themain aetiological cause but occupational environment andunderlying genetic makeup also play significant roles indeveloping chronic lung disease These diseases are usuallyassociated with development of airway inflammation airwayhyperresponsiveness (AHR) and mucus overproductionCOPD refers to conditions characterised by chronic orrecurrent obstruction of air flow including chronic bronchitisand emphysema Chronic bronchitis results from recurrentepisodes of acute bronchitis or persistent and noninfec-tive irritation of bronchial mucosa This disease is usuallyassociated with emphysema a condition characterised bypersistent dilatation of air spaces and destruction of theirwalls [7] Asthma is characterised by a variety of features

including reversible airway obstruction airway inflamma-tion and increased airway responsiveness to both physicaland chemical stimuli [8]

2 Rationale for Using Animal Models toStudy Lung Disease

Lung diseases are extremely prevalent worldwide thus thedevelopment of prevention strategies and new treatmentmethods to reduce the worldwide burden and increasepersonal quality of life is crucial As for any other diseaseclinical investigation and epidemiological studies are neededto advance knowledge and improve disease management [9]A biological model is needed to study the mechanisms thatunderlie the development of lung disease at both cellular andmolecular levels

Human models of ALI have been reported in severalcase studies Human models were used in a clinical trial todevelop and test a novel therapeutic agent targeted to cureALIARDS [10] However there are drawbacks to use humansas research subjects Humans can suffer from other diseaseduring the experimental period and there are risks associatedwith experimental procedures (eg inducing a low level ofinflammatory response) Therefore healthy volunteers mustbe thoroughly educated about the potential risk of participat-ing in a given study Various ethical and pathological issuesalso limit the use of humans as an in vivomodel

Thus animals are preferable as an experimental modelfor modelling the human respiratory system They providean experimental setting that allows researchers to study theinteraction between the immune system and a functioningrespiratory system Choosing an ideal animal model for usein lung disease studies must follow several criteria The mostimportant is the ability of the model to reproduce the princi-pal aspect of human lung disease The model also should be

BioMed Research International 3

able to imitate the sensitised human lung conditions in termsof the nature of the injury and any disease-induced changesthat occur at biological physiological and pathological levels

For example when modeling ALIARDS in an animalmodel the model should be able to mimic the sensitisedhuman lung condition which includes injury at the alveolar-capillary membrane neutrophil-induced inflammation andincreased permeability of pulmonary oedema (Table 1) Tosimulate human ALIARDS the animal model also shouldreproduce the acute lung injury to the epithelial and endothe-lial barriers in the lungs and the acute inflammatory responsein the air spaces [2]

For chronic lung diseases such as asthma an effectivemodel should reproduce the principal aspects of humanasthma which include immunoglobulin E (IgE)-mediatedsensitivity to antigens acute bronchoconstriction increasedairway resistance chronic inflammation of the airwaysTh2 cytokine production eosinophilia late phase airwayobstruction enhanced mucus secretion decreased mucocil-iary clearance airway wall remodelling and smooth musclehyperplasia [11] In addition an ideal animal model musthave the ability to develop disease and evolve injury over aprolonged period of time [12]

In order to mimic the pathophysiology of a humandisease a large-sized animal is more suitable than a small-sized animal because the complexity of its organ structureis more similar to that of humans Moreover observationof disease development is much easier in larger animalsDifferent animal models have been used to study lungdisease includingmice guinea pigs and rabbits Each animalpossesses certain advantages and disadvantages as a model oflung disease Below are descriptions of the pros and cons ofvarious animals for use in studying lung disease particularlyasthma (Table 2)

21 Mouse The mouse is an ideal model for studyingmost diseases because we have a detailed understanding ofmouse genetics [20 21] The mouse has become the mostpopular animal for modelling allergic airway responses suchas asthma IgE is the primary allergic antibody in micewhich is similar to the situation in humans therefore thisspecies is thought to be suitable for investigation of humoralimmune factors in the development of allergic airway disease[8 9] Moreover numerous immunological reagents andtools for use in mouse studies are available and they offerthe opportunity to explore detailed mechanisms of allergicreactions [8 9 22] In addition the availability of knock outor transgenic species makes mice a preferred model for anystudy related to genemanipulation [8] Furthermoremice arerelatively cheap and easy to breed and have a short gestationalperiod thus allowing large studies to be conducted [8 22]

Despite these advantages mice and humans have consid-erable physiological differencesThemost relevant differencesfor lung disease studies are differences in lung anatomyand airway musculature In the mouse vasculature is theprimary target of anaphylactic response whereas the lung isthe primary target for anaphylactic response in humans [9]In addition the poorly developed airwaymusculature inmice

Table 1 Characteristics of human lung injury

Clinical features

Acute onsetDiffuse bilateral alveolar injuryAcute exudative phaseRepair with fibrosis

Physiological changes

VQ abnormalitiesSevere hypoxemiaDecreased complianceImpaired alveolar fluid clearance

Biological changes

Increased endothelial and epithelialpermeabilityIncreased cytokine concentration in lungsProtease activationCoagulation abnormalities

Pathological changes

Neutrophilic alveolar infiltratesIntra-alveolar coagulation and fibrindepositionInjury of the alveolar epithelium withdenudation of the basement membrane

suggests that developing a physiological model of pulmonaryresponsiveness is inappropriate in this species [9]

Plasma exudation is a cardinal sign of bronchial asthmaand allergic rhinitis [23] In humans numerous plasma-derived inflammatory- repair- leukocyte- and growthfactor-active proteins irrespective of size are distributedthroughout the airway tissues In contrast Gelfand [23]reported that mice showed little plasma exudation espe-cially in the late phase response even after allergy loadingIn sensitised mice the inflammatory response to antigenchallenge usually results in a massive influx of inflammatorycompounds dominated by eosinophils into the airways [22]From an immunological point of view eosinophils rarelydegranulate inmousemodels of asthma whereas they readilydegranulate in humans Because both plasma exudation andeosinophil degranulation may correlate with disease severityit appears that asthma-like symptoms aremore or less lackingin the allergicmousemodel [23] In addition themast cells ofmice and rodents generally release serotonin but this is notthought to play a role in human asthma For these reasons themouse model is not suitable for studying asthmatic diseaseAllergic mice also do not exhibit spontaneous AHR andsmooth muscle hyperplasia is not easily demonstrated [8]

22 Guinea Pig Because guinea pigs are small and docile ani-mals for over 100 years they have been the most widely usedtest systems for contact hypersensitivity to chemical irritantsand proteins Some researchers may assume guinea pigs as anideal model to study asthmatic disease because they developwell-characterised early and late phase airway responses toallergen challenge following sensitisationThis allowsmecha-nistic investigation of each reaction as well as the relationshipbetween the two responses [9 20]The associated pulmonaryinflammatory responses which involve both eosinophils andneutrophils are consistent with asthma [8]


Table 2 Advantages and disadvantages of various animal models for studying lung disease particularly asthma [8 9]

Animal Advantages Disadvantages

Mouse

IgE is the major anaphylactic antibody Do not exhibit spontaneous AHRNumerous inbred strains Limited airway musculatureNumerous immunological reagents Lung anatomy differencesSmall relatively inexpensive Do not easily demonstrate smooth muscle hyperplasia

Guinea pigThe lung is the primary organ of anaphylaxis IgG1 is the major anaphylactic antibodyShow early and late phase airway responses Shortage of inbred strainsSmall docile animals inexpensive Few species-specific reagents

Rabbit

Phylogenetically similar to humansThe lung is the major target organIgE is the major anaphylactic antibodyLarge enough to study for lung mechanicsDemonstrate both early and late phase airway responses

Neonatal immunisation required for developing latephase airway responseFew species-specific reagentsFew transgenic species availableGene sequences are not well mapped

This species produces both IgG1 and IgE antibodies withIgG1 being more dominant than IgE From an immuno-logical point of view this presents difficulties in studiesof mechanisms of humoral responses to allergens [9] Inaddition mechanistic studies especially studies involvinggenetics using the guinea pig model are limited due to thelow number of inbred strains available A limited number ofspecies-specific reagents is available thus making it difficultto identify and isolate particular cell types such as lymphocytesubsets

23 Rabbit The rabbit is phylogenetically closer to primatesthan are rodents Moreover the rabbit provides an animalmodel that resembles humans in that the lung is the targetorgan for anaphylactic response This species demonstratesboth early and late phase airway responses thus allowingmechanistic investigation of each reaction and the relation-ship between themThe latterrsquos associationwith inflammationis thought to be of great importance in the development ofasthma [9] In order for rabbits to develop late phase airwayresponse neonatal immunization is required [7]

From an immunological point of view rabbits produceIgE as the primary anaphylactic antibody The presence ofIgE is necessary to initiate antigen-induced late responses inthe rabbitrsquos lungs [24] Rabbits are easy to handle and readilyavailable making them a good model for investigating lung-related disease In addition rabbits are large enough to allownonlethal monitoring of physiological changes

Despite the many advantages of the rabbit as an animalmodel this species has not been widely used probably due tolimitations in terms of cost and reagent availability The costof the animal itself as well as the space required to house itmakes the rabbits more expensive to use than smaller speciessuch as guinea pigs or mice [7] In addition gene sequencesfor rabbits are not well mapped at this time

Due to the low numbers of knock out or transgenicspecies and the lack of rabbit-specific reagents mechanisticrabbit studies are also limited particularly those involvinggenetics [7] A few transgenic rabbits are available but moststudies involving the use of transgenic rabbits have focussedon pulmonary cardiovascular and metabolic issues [25]

This paper focuses on the use of rabbits in modeling bothacute and chronic lung diseases and as a model for stem celltherapyWhen considering the use of rabbits there are severaldistinct components that need to be considered especiallyanatomical structure of the rabbit airway and the ability ofthe rabbit to imitate human lung disease as much as possible

3 Background on the Use of the Rabbit asa Disease Model

Mimicking the human condition in the animal model isdecisive and critical to obtain accurate and comparable results[26] Thus it is challenging to choose the right animal modelsystem One of the most crucial aspects to consider whenchoosing the suitable model is anatomical structureThe lungis a sac organ that functions to contain and allow exchangeof respiratory gases It is a complex structure consisting oftracheobronchial branches and cellular parenchyma whichdrive the dynamic physiological mechanism of the lungThus these anatomical features of the lungmust be comparedbetween humans and rabbits to evaluate the ability of therabbit animal model to recreate human chronic lung disease(Table 3)

31 Respiratory Bronchioles Respiratory bronchioles (RBs)are important airway anatomical structures that differbetween species RBs are partially alveolarised airwayslocated between the terminal bronchioles and the alveolarducts Absence of RBs enable the clearance of insolubleparticles from alveolar ducts directly to terminal bronchiolesThese were proved to be efficient Clearance failure results inthe pathological condition Unlike rabbits humans possessRBs which evidently play a role in the development ofemphysema and fibrosis [27]

32 Tracheobronchial Capillary Bed The airway mucosa isa relatively highly vascularised structure In a pathologicalcondition like asthma vascular permeability changes con-tribute to mucosal thickness At this point inflammatorycells chemical mediators and plasma protein migrate from


Table 3 Anatomical comparison between the human and rabbit lung

Anatomical structure Human Rabbit Reference(s)Respiratory bronchioles Present Absent [13]Tracheobronchial capillary bed 7 capillariesmm 5 capillariesmm [14 15]Branching pattern More symmetrical 25 generations Less symmetrical 32 generations [16ndash18]Lung expansion rate 26 folds 20 folds [16 17]Mucus producing cells Submucosal glands and goblet cells Only goblet cells [19]

blood vessel to the interstitial tissue Accumulation of theseinflammatory substances leads to the oedema and mucosalthickness The arrangement of the vascular circulation con-sists of the submucosal plexus interconnected with an outeradventitial plexus of vessels which is located outside thesmoothmuscle and bronchial cartilage [28] Rabbits have fivecapillaries per mm whereas humans have seven capillariespermm [29] Compared to other species humans and rabbitshave a minimal capillary network

33 Branching The lower respiratory airway begins withbifurcation of the trachea into two bronchi one leading toeach lung Both main bronchi further subdivide into theirrespective lung subsmenental lobar Further branching intobronchioles occurs with airway trees ending in terminalbronchioles Terminal bronchioles are connected to alveolarducts where gas exchange took place [30] Humans and rab-bits have 25 and 32 generations of airway branching respec-tively [13 31] and they exhibit a similar symmetrical branch-ing pattern [14] Each of the main branches divides intotwo asymmetrical daughter branches Twodaughter branchesthat have an identical diameter are referred to as dichoto-mous whereas an imbalance of the diameter is referred toas monopodial Branching in humans is almost perfectlydichotomous whereas in rabbit the diameter of the airwaybranches is monopodial The branching pattern may influ-ence the distribution and deposition of air and particles [15]

34 Lung Volume Changes in lung volume are related tolung dysfunction and disease impacts on lung mechanicsgas exchange respiratory muscle function the sensation ofdyspnoea and tolerance to maximum exercise Monitoringof lung volume is crucial when conducting an experiment tostudy a pathological condition In rabbits and humans lungexpansion exhibits similar growth from birth to adulthoodwith a 20-fold and 26-fold increase respectively [16 17]

35 Airway Epithelial Layer The airway is a pipe-like struc-ture that is lined by a pseudostratified epithelial cell layer andeach cell plays a critical role in maintaining the passage of airAs a group these cells act as a protective barrier and defencemechanisms against foreign air particulates [32] Goblet cellsciliated cells and basal cells line the epithelial layer of thelarge proximal airways In the more distal airways gobletcells gradually are replaced by Clara cells The compositionof the respiratory epithelial cells varies among species Celldensity is one of the prominent differences between humansand rabbits

36 Mucus Producing Cells In the airways mucus is pro-duced by goblet cells and submucosal glands It is a maincomponent of the mucus gel layer which is located atthe apical epithelium Mucus consists of water electrolytesantimicrobial substances anti-inflammatory cells and manyother compounds Thus mucus serves as a neutraliser offoreign materials Mucus overproduction is a main hallmarkof pathological conditions as it leads to obstruction of theairways This is due to increased number of goblet cellswhich often called hyperplasia andmucus production rate Inhumans submucosal glands are distributed along the airwayand are concentrated between the cartilaginous rings [19]thus making goblet cells are abundantly seen in the epitheliallayer [19] In contrast rabbits do not have submucosal glandsand goblet cells appear to be less abundant compared tohumans [19] Despite the absence of submucosal glandsthe rabbit has been used in studies specifically focussed onchanges in goblet cells that occur in response to a pathologicalcondition [33]

37 Use of the Rabbit as a Model for Lung Disease Therabbit is phylogenetically closer to humans than are rodentsBecause of the anatomical physiological genetic and bio-chemical similarities between rabbits and humans thisspecies is preferentially used in pulmonary cardiovascularand metabolic studies including those of airway obstructivedisease embolic stroke arteriosclerosis cholera and cysticfibrosis As a classical experimental animal model rabbitsalso are used for drug screening tests antibody productionand the production of therapeutic proteins

4 Remodelling of Lung Disease in Rabbit

There are several distinct components that need to beconsidered when modelling lung disease in animal modelsThese include protocols for sensitisation and methods formeasuring the extent of disease such as lung inflammationAHR and others

41 Sensitisation Method A number of allergens have beenused in animal models of lung disease especially asthmaIn the 1970s Pinckard et al [18] introduced the method ofintraperitoneal (ip) administration of antigen in combina-tion with adjuvant to neonatal rabbits They injected solublebovine serum albumin in conjunction with Corynebacteriumparvum adjuvant to newborn rabbits within the first 24 h oflife and this continued every week according to their study


design The regime resulted in the production of antigen-specific IgE antibodies

This protocol has subsequently been modified and vari-ous antigens have been used to sensitise animals especiallyrabbits Among them are ovalbumin (OVA) lipopolysac-charide house dust mite (HDM) and ragweed pollen[7] Shampain et al [24] conducted a study in whichthey sensitised rabbit neonates with Alternaria tenuis asthe allergen This regime leads to the development ofearly and late airway responses to acute antigen challengeDouglas et al [34] performed a study to determine theeffect of long-term exposure of sensitised rabbits to envi-ronmental pollutant gases in conjunction with the aetiol-ogy of asthma They sensitised different rabbits with twotypes of allergen A tenuis and HDM In another studyresearchers used a combination of allergens in a single animalmodel animals were sensitised either with single doubleor triple allergens (HDM ragweed Aspergillus fumigatus)[34]

OVA has been widely used to sensitise and challenge hostanimals As OVA is readily available and can be easily manip-ulated via diet such that animalrsquos immune systemhas not beenexposed to OVA before sensitization [22]The OVAmodel ofairway inflammation is usually characterised by high levelsof OVA-specific IgE and eosinophils a T-cell predominantbronchial inflammatory response and the development ofAHR [20] which are similar to characteristics of asthma inhumans

A typical sensitisation protocol usually involves inject-ing the allergen with adjuvants into the animal Adjuvantssuch as aluminium hydroxide (alum) are responsible forinducing strong and sustained sensitisation promoting thedevelopment of aTh2 phenotype by the immune system andpromoting production of allergen-specific IgE and IgG1 [2235] Other adjuvants that have been used in sensitising animalmodels include heat-killed Bordetella pertussis raisins andadjuvant mixes such as Freundrsquos complete adjuvant [22]However this adjuvant mix is known to promote a moreTh1-biased response which is not comparable with characteristicsof human asthma

Conrad et al [36] compared experimental asthma phe-notypes between adjuvant and adjuvant-free protocols ininducing allergic airway inflammation in murine The sen-sitisation protocol involved injection of antigens using twodifferent routes (ip and subcutaneous (sc)) They foundthat the sc adjuvant-free protocol resulted in significantlyhigherOVA-specific IgE and significantly lowerOVA-specificIgG1 levels than the ip adjuvant protocol Differences inthe levels of IgE and IgG1 between these two protocolsresulted from the adjuvant itself Alum participates in thegeneration of humoral immunity [36] thus it promoteshigh production of OVA-specific IgG1 antibodies in com-parison to an adjuvant-free protocol Conrad et al [36]concluded that the sc adjuvant-free protocol generateda phenotype that was similar to the standard OVA ipadjuvant protocol used in the majority of studies How-ever different adjuvants and their use or omission provideoptions for researchers when designing sensitisation proto-cols

42 Lung Function To investigate physiological parameterssuch as lung function it is very important to work withindividual animals that can undergo repeated assessmentduring the time course of the study Thus it is importantto use an animal for which longitudinal responsivenesscan be investigated as this would occur clinically in theinvestigation of human subjects with asthma For this reasonthe rabbit is a better model for investigating lung functioncompared to other animals Rabbits can act their own controlwhen measuring lung function as repeated measurementscan be made in rabbits using an endotracheal tube andoesophageal balloons This technique is clearly impossiblein smaller animals such as rats and guinea pigs for whichinvasive surgery is needed to perform the investigation Inaddition anaesthetised rabbits remain breathing throughoutthe course of the experiment thereby excluding the need formechanical ventilation which is required for smaller animals[7]Thus the rabbitmodel offers advantages for lung functionstudies

43 Airway Hyperresponsiveness AHR can be defined as anincrease in sensitivity and reactivity of the airways in responseto physical and chemical stimuli [37]This AHR is consideredto be the hallmark of asthma and greater AHR has been cor-related with increased disease severity [38] Sensitised rabbitneonates have shown an enhanced responsiveness to variousstimuli including histamine methacholine and adenosine51015840 monophosphate (AMP) [7] AMP causes a marked dose-related bronchoconstriction that may occur within minutesto an hour if inhaled by atopic asthma patients AHR toinhaledAMPmay reflect the severity of airway inflammationand this phenomenon has been demonstrated in variousanimal species In 1996 El-Hashim et al [39] reported bron-choconstriction in response to inhaled adenosine in allergicrabbits and it appeared to be mediated by the activationof A1receptors In another study using the allergic rabbit

model Obiefuna et al [40] described the role of the A1

adenosine receptor antagonist L-97-1 in reducing antigen-induced early and late responses and the allergen-inducedhyperresponsiveness to inhaled histamine and adenosine

44 Early and Late Phase Airway Responses Rabbits candevelop both early and late phases of airway response butneonatal immunisation is required for the late phase responseto be developed Herd et al [41] reported that neonatalrabbits immunised within 24 h of birth [41] exhibited manyfeatures of human asthma including AHR in response toinhaled antigen acute and late phase airway obstruction [24]pulmonary eosinophil and lymphocyte recruitment [41 42]and production of IgE antibodies [24 43] This clearly showsthe ability of the rabbit to develop disease from the point ofbeing sensitised through adulthood thus making this speciesideal as a model for human asthma

Theoretically bronchial allergen challenge in sensitisedatopic asthmatics will lead to both acute and late phaseairway obstruction The acute phase occurs as a result ofcontraction of the smooth muscle of the airway whereas thelate phase occurs due to inflammation Studies have shown


that adult rabbits that have been neonatally immunised toan antigen undergo both early phase and late phase airwayobstruction in response to acute exposure of the airways toan aerosolised antigen [24 44] This finding is far differentfrom what is seen in rats Even though rats demonstrateboth acute and late phase airway constriction the severityof bronchoconstriction depends on the antigen used and iscorrelated with the titre of specific IgE antibody in the serum[45] Bronchoconstriction is rarely provoked if the IgE titreassessed using the passive cutaneous anaphylaxis assay islower than 16 [9]

In addition to the early response allergen challenge ofatopic subjects with asthma leads to AHR 24 h later [7]The late response usually is associated with an increasedresponsiveness of the airways to various stimuli includinginhaled histamines and methacholine Neonatally immu-nised adult rabbits exhibit similar hyperresponsiveness after24ndash48 h following allergen challenge In contrast mice do notdemonstrate spontaneous AHR as they fail to develop anairway constrictive response to histamine [8] In particularit is unclear whether mice are capable of exhibiting a physio-logical late phase constriction in the lung [46 47]

45 Airway Inflammation Inflammation plays a key role inhuman asthma Asthmatic patients usually exhibit signs ofinflammation such as infiltration of inflammatory cells intolung tissues high levels of IgE and IgG1 in sera thickeningof airway epithelial tissue and excessive mucus secretiondue to goblet cell hyperplasia and hypertrophy of mucousglands [48] In the case of allergic rabbits following allergenchallenge bronchoalveolar lavage (BAL) may be performedto assess the extent of cell infiltration Typically lung lavageis performed using saline via an endotracheal tube Thecollected BAL fluid is used to determine both total anddifferential cell counts [7] In addition the pathology ofasthma in allergic rabbits can be observed histologically

The most important pathological change that occurs inthe lung during injury is the infiltration of neutrophils intothe lung During the hypersensitivity response inflammatorycells are recruited into affected sites Neutrophils are the firstcells to be recruited to the site of inflammation and theyhave potent antimicrobial armour which includes oxidantsproteinases and cationic peptides [49] Activated neutrophilsand alveolar macrophages secrete inflammatory mediatorsthat disrupt epithelial fluid transport and impair surfactantproduction by alveolar type II cells [10] Several studies ofthis process have been conducted using animal models suchas neutrophil assessment during ALIARDS onset in rabbits[50] In a study using the rabbit as an in vivomodel Folkessonet al [51] showed that neutrophil recruitment into the areaof inflammation was highly regulated by the presence ofinterleukin-8 (IL8)

5 The Potential Use of the Rabbit Model inDrug Screening Tests

Animals have been widely used for drug screening tests (egin order to predict the safety and effectiveness of a drug

before it is used clinically)The allergic rabbit model has beenextensively used in assessing various antiasthmatic drugsIn general the allergic rabbit is sensitive to similar drugsas human patients with asthma Clinically isoprenaline hasbeen reported as not being able to reverse the late reaction inasthmatic patients once it developed [24] In the preclinicalsetting this drug also is unable to induce any effect on theincrease in airway resistance or the decrease in dynamic lungcompliance in the allergic rabbit model [7]

Administration of sodium cromoglycate in the allergicrabbit prior to allergen challenge was shown to inhibitboth early and late phase responses This finding has alsobeen clinically shown in asthmatic patients as discussedby Altounyan [52] Administration of xanthines in humansinhibits the development of the late response following aller-gen challenge In rabbits aerosolised theophylline was shownto inhibit both the early and late phase of bronchoconstrictioninduced by antigen as well as AHR [7]

Peptide leukotrienes can induce many features of asthmain both humans and experimental models including airwayobstruction mucus secretion increased vascular permeabil-ity inflammatory cell infiltration and AHR [7] The effect ofPF 5901 a leukotriene synthesis inhibitor and LTD

4antag-

onist had been tested in rabbits and it has been reportedthat the drug had no effect on acute bronchoconstriction oreosinophil infiltration Nevertheless AHR was inhibited andlevels of LTD

4were reduced following the antigen challenge

[41]Corticosteroids can be used to treat ALIARDS in asthma

patientsThe administration of the corticosteroid budesonidein sensitised rabbits resulted in marked anti-inflammatoryactivity and inhibited antigen-induced AHR and bron-chodilator responses [53] Budesonide also can inhibit theearly and late responses as well as eosinophil infiltration [7]

6 The Rabbit as a Model forCell Therapy Research

61 Airway Epithelium Injury Various methods have beendeveloped to target the damage to particular regions of theairway due to inhalation or iatrogenic causes [54] Inhalationof foreign substances into the lungs can block the airwayand also interfere with gas exchange directly by physicalobstruction or indirectly by provoking acute bronchospasmor delayed inflammation Iatrogenic injury due to intubationcan cause upper airway obstruction from oedema Bothinjuries caused disruption of cells that can be classified intofour categories (i) reversible injury to the airway which willheal and return to normal condition once the damage isremoved (ii) exfoliation of individual cells with the majorityof nonciliated columnar and basal cells left intact (iii)desquamation of groups of cells but with the basal cell layerleft intact and (iv) desquamation of cells including the basalcells [55]

62 Repair Process following Injury Under normal circum-stances the damaged epithelium is able to repair itself rapidly


Immediately after injury occurs the airway epithelium initi-ates a repair process in order to restore the integrity of thebarrier [56] The cells that participate in wound healing andfunctional regeneration of the epithelium in both humansand rabbits are the epithelial basal cells of the trachea andbronchi Clara cells of the bronchioles and alveolar type II(ATII) cells [57]

The regeneration process is a complex phenomenon thatquickly starts after the lesion occurs These cells can rapidlychange their structure and function in order to adapt tochanges in the local environment or to repair the epitheliumafter injury In vitro studies of human airway epithelium cellrestitution have shown that the regenerative process to repairthe airway epithelium involves dedifferentiation migrationof neighboring epithelial cells to cover the denuded areaproliferation of progenitor cells to restore cell numbers andredifferentiation to restore the function of epithelial cells[55 57ndash59]

63 Stem Cells and Progenitor Cells Stem cells are definedas unspecialized cells that have the remarkable potential todevelop into varying types of cells As an internal repairsystem these cells can replenish injured cells For cells to fallunder the definition of stem cells they must have the abilityof unlimited self-renewal to produce progeny that is exactlythe same as the originating cell and they must be able to giverise to a specialized cell type that becomes part of the organtargeted [60] Most organs contain their own small reservoirof stem cells also called progenitor cells which are recruitedto start dividing and replace cells that have died duringnormal aging or to repair small areas of damage [61] Stemcells also can differentiate into progenitor cells which arelineage-specific precursors to a more restricted developmentpotential [62] Several potential sources of progenitor cells forairway epithelium have been identified and are divided intotwo groups endogenous stem cells and exogenous stem cells[63]

64 Endogenous Stem Cell Endogenous stem cells arealready present in the respiratory tract In both rabbits andhumans the basal cells are the primary stem or progenitorcells in the airway and the ability to differentiate into othertypes of cells such as secretory or ciliated cells can be detectedby using cell proliferation markers [64] Clara cells which arelocated in the bronchioles are believed to be capable of return-ing to proliferation after injury [65] In the gas-exchangingregion of the lung ATII cells are the progenitor cells andcan differentiate into ATI-like cells [66] Collectively thesestudies have shown that stem or progenitor cells are recruitedinto the injured area The cell proliferation and phenotypicdifferentiation also lead to recovery of epithelium function

65 Exogenous Stem Cell The repair ability possessed byendogenous lung epithelial progenitor cells is often insuffi-cient as the natural repair capacity appears to diminish withage [67]Direct interfacewith the outside environmentmakesepithelial cells inside the lungs susceptible to potential toxicagents and pathogens thus they must be able to respond

quickly and effectively to cellular damage [68] Although thedamage can be rapidly and completely repaired by progenitorcells these cells are usually present in insufficient numbers tocompensate for extensive damage [61] The purpose of stemcell transplantation is to replace damaged or lost cells in anorgan or tissue Autologous or allogeneic cell transplantationin experimental models has shown that these cells canengraft in the lung and differentiate into mature epithelialphenotypes [69 70] and thus increase the cellular responseto injury [71]

66 Cell-Based Therapy Cell-based therapy has attractedtremendous interest recently The concept of regenerativemedicine using cells to repair tissues represents the poten-tial to develop alternative therapeutic strategies that mayultimately play a major role in the treatment of a numberof diseases [72] There are many possibilities for cell-basedtherapy depending on the disease itself Given the abilityof regeneration cell-based delivery offers new potential fortreating diseases such as COPD and asthma Much still isneeded to be learned about their characteristics manipula-tion safety and application of these cells for effective cell-based therapy to treat diseases Moreover for cells to besuccessfully used for human treatment precise delivery andtargeting of the cells to the site of the diseasemust be ensured

An overview of in vivo studies of cell-based therapy thatused the rabbit as an animalmodel is provided belowMost ofthese studies indicated that this kind of treatment is feasiblesafe and likely to be applicable in clinical trials in the nearfuture

67 Bone-Marrow-Derived Stem Cells Adult stem cells suchas hematopoietic and mesenchymal stem cells (MSCs) arefound inmature bonemarrow tissues Plasticity of adult stemcells means that they can generate lineages of cells that aredifferent from their origin Thus these cells can be used fororgan regeneration and for cellular repair in various animalspecies as well as in humans [72] The ability to differen-tiate into various mesodermal cells makes MSCs the mostcommonly used adult stem cells in cell-based therapy MSCscan differentiate not only into mesenchymal lineage cells butalso into endothelium and endoderm in vitro [73] HoweverMSCs also can be organ specific as populations isolatedfromvarious sourcesmight be functionally different althoughmorphologically similar For example MSCs isolated fromthe umbilical cord do not have the same ability to give riseto osteoblasts chondrocytes and cardiomyocytes as bonemarrow-derived MSCs [74] Three basic mechanisms havebeen proposed to explain howMSCs can repair tissue injury(a) creation of amilieu that enhances regeneration of endoge-nous cells (b) transdifferentiation or (c) cell fusion [75ndash77]Because of their potential use in regenerative medicine thetherapeutic potential of MSCs has been explored throughautologous or allogeneic transplantation in patients throughlocal delivery or systemic infusion [78]

Zhu et al [79] studied the effect of MSC engraftmenton vascular endothelial cell growth factor (VEGF) in rabbitlung tissue plasma and extravascular lung water at an early


stage of smoke inhalation injury MSCs inhibit the acti-vation and proliferation of immune cells by secreting aninhibitory factor to reduce the production of factors thatinduce the production of VEGF VEGF itself participates inan inflammatory reaction formation of pulmonary oedemaand aggregation of neutrophilic granulocytes in the lungThus the engraftment of MSCs had a protective effect onsmoke inhalation injury through regulation of the local andsystematic VEGF and reduction of lung water content

Haematopoietic stem cells are progenitors for several celltypes such as endothelial cells epithelial cells myocytes andneurons and they also are able to reduce lung fibrosis [80]Bone marrowmononuclear cells (BMnCs) which are knownto contain both haematopoietic cells and MSCs were shownto have therapeutic potential for acute lung injury [81] andlung fibrosis [82 83] BMnCsmay improve lung injury due tobone marrow cell differentiation into epithelial and endothe-lial cells [81] Yuhgetsu et al [84] used autologous BMnCs tomitigate elastase-induced pulmonary emphysema in rabbitsThe cells were transplanted via the left and right mainbronchi 4 weeks after administration the rabbits showedsignificantly better pulmonary function and smaller alveolarairspaces This inhibition of progression of emphysema wasdue to the BMnCsrsquo ability to attenuate inflammation MMP-2 expression and apoptosis while enhancing alveolar cellproliferation

68 Endothelial Progenitor Cells Endothelial progenitor cells(EPCs) can be isolated from peripheral blood or bonemarrow They react to physical and chemical stimuli withinthe circulation and regulate homeostasis vasomotor toneand immune and inflammatory responses [85] CirculatingEPCs play important roles in angiogenesis [86] and repairof injured endothelium [87] Circulating EPCs are mobilizedfrom the bonemarrow to peripheral circulation by cytokinesgrowth factors and ischemic conditions during endothelialinjury [86]

Lam et al [88] studied circulating EPCs as a potentialtherapeutic treatment for ALI One week after culturingEPCs ALI was induced in rabbits by oleic acid and autolo-gous EPCs were then transplanted intravenously The EPCspreserved pulmonary endothelial function and maintainedthe integrity of the pulmonary alveolar-capillary barrier Theparacrine effect of EPCs due to secretion of growth factorscontributed to the overall beneficial effect on endothelialrepair Another study using circulated EPCs from peripheralblood mononuclear cells was designed using the rabbit as amodel He et al [89] demonstrated that after 4 weeks trans-plantation of autologous EPCs enhanced endothelialisationand improved endothelial function of the denuded carotidartery

69 Tissue Engineering Tissue engineering involves creatingwhole organs or parts of organs to replace and repair damagedtissues Constructing three-dimensional tissues from two-dimensional cell layers outside the body is a major challengethat requires very specific knowledge and skills The processinvolves three important steps First the desired cells which

can be derived from stem cells must be obtained Howeverthe stem cells must differentiate into the targeted cells firstSecond the cells need a structure or scaffolding on whichto attach grow and carry out their specialized function(eg producing bone or cartilage proteins) Last the three-dimensional tissue-engineered organ part should have anadequate supply of oxygen and cell nutrients [61]

Tissue engineering presents a promising technique tocreate a functional organ substitute In one study a tissue-engineered trachea fabricated from fibrin gel and autologouschondrocytes was transplanted into rabbits successful regen-eration and functional restoration of ciliated epithelial cellson the operated site were achievedwithout graft rejection andinflammation [90] Rabbits also have been used to study air-way stem cell biology and lineages using purified or enrichedspecific epithelial cell types to reconstitute denuded trachealgrafts Inayama et al [91] demonstrated the ability of rabbitbasal cells to repopulate epithelium containing basal ciliatedand goblet cells after reconstitution of a tracheal xenograft

7 Conclusion

Overall the rabbit is a usefulmodel for studying lung physiol-ogy and pathophysiology The rabbit as a model offers betterunderstanding of lung structure than smaller animal modelsThe rabbit model permits investigation of chronic aspects ofdisease because it can act as a control by allowing multiplemeasurements to be made at different time pointsThe abilityof the rabbit to develop diseases once sensitised with severalstimuli at birth provides opportunities to investigate the riskfactors that contribute to allergic diseaseThemost importantaspect of the rabbit model for lung disease is its similarities tohumans in terms of asthma development thus it provides anideal basis studying the effects of novel asthma therapies [7]

Remarkable progress has been achieved in the study ofstem cells as avenues to provide clinical deliverables Stemcell therapy represents a fascinating new approach for themanagement and repair of injuries Recent in vivo studiesusing rabbit models have shown the feasibility of autologousand allogeneic cell therapy However current studies are inthe early stages and there is still much to be learned suchas how to enhance production survival and integration oftransplanted cells prior to realistically contemplate clinicalstrategies

Although the rabbit as a model has several limitationsthese limitations can be overcome with modification andimprovement It is important to remember that a rabbit orany other animal model cannot be considered as a surrogatefor human disease Instead such models should be seen asan important opportunity to generate and test hypotheses ina simple and controlled system The clinical relevance of thefindings in rabbits and other species can only be determinedin human studies [8]

Acknowledgments

The publication of this paper has been supported by the Uni-versiti SainsMalaysia (USM)ResearchUniversity (RU)Grant


Schemes (1001CIPPT811204 and 1001CIPPT813059) E-ScienceFund Scheme Grant (305CIPPT613224) by Min-istry of Science Technology and Innovation (MOSTI) andUSM Short Term Grant Scheme (304PPSP61311018 and304CIPPT61312001)

References

[1] A Dushianthan M P W Grocott A D Postle and R CusackldquoAcute respiratory distress syndrom and acute lung injuryrdquoPostgraduateMedical Journal vol 87 no 1031 pp 612ndash622 2011

[2] G Matute-Bello C W Frevert and T R Martin ldquoAnimalmodels of acute lung injuryrdquo American Journal of Physiologyvol 295 no 3 pp L379ndashL399 2008

[3] T Geiser K Atabai P H Jarreau L B Ware J Pugin and MA Matthay ldquoPulmonary edema fluid from patients with acutelung injury augments in vitro alveolar epithelial repair by an IL-1120573-dependentmechanismrdquoAmerican Journal of Respiratory andCritical Care Medicine vol 163 no 6 pp 1384ndash1388 2001

[4] M A Matthay and G A Zimmerman ldquoAcute lung injury andthe acute respiratory distress syndrome four decades of inquiryinto pathogenesis and rational managementrdquo American Journalof Respiratory Cell andMolecular Biology vol 33 no 4 pp 319ndash327 2005

[5] B Moldoveanu P Otmishi P Jani et al ldquoInflammatory mecha-nisms in the lungrdquo Journal of Inflammation Research vol 2 pp1ndash11 2008

[6] A Z Latahir and B Yahaya ldquoBlinded brushing technique as anovel method to inflict injury on rabbit tracheal airway epithe-lium structurerdquo Journal of Animal and Veterinary Advances vol11 no 20 pp 3772ndash3775 2012

[7] S Keir and C Page ldquoThe rabbit as a model to study asthma andother lung diseasesrdquo Pulmonary Pharmacology and Therapeu-tics vol 21 no 5 pp 721ndash730 2008

[8] Y S Shin K Takeda and E W Gelfand ldquoUnderstandingasthma using animal modelsrdquoAllergy Asthma and ImmunologyResearch vol 1 no 1 pp 10ndash18 2009

[9] M H Karol ldquoAnimal models of occupational asthmardquo Euro-pean Respiratory Journal vol 7 no 3 pp 555ndash568 1994

[10] A G Proudfoot D F McAuley M J D Griffiths andM HindldquoHumanmodels of acute lung injuryrdquoDMMDiseaseModels andMechanisms vol 4 no 2 pp 145ndash153 2011

[11] P C Fulkerson M E Rothenberg and S P Hogan ldquoBuilding abetter mouse model experimental models of chronic asthmardquoClinical and Experimental Allergy vol 35 no 10 pp 1251ndash12532005

[12] M Ostroukhova C Seguin-Devaux T B Oriss et al ldquoTol-erance induced by inhaled antigen involves CD4+ T cellsexpressing membrane-bound TGF-120573 and FOXP3rdquo Journal ofClinical Investigation vol 114 no 1 pp 28ndash38 2004

[13] R Ramchandani J H T Bates X Shen B Suki and R S Tep-per ldquoAirway branching morphology of mature and immaturerabbit lungsrdquo Journal of Applied Physiology vol 90 no 4 pp1584ndash1592 2001

[14] R B Schlesinger and L A McFadden ldquoComparative mor-phometry of the upper bronchial tree in six mammalianspeciesrdquo Anatomical Record vol 199 no 1 pp 99ndash108 1981

[15] H C Yeh R F Phalen and O G Raabe ldquoFactors influencingthe deposition of inhaled particlesrdquo Environmental HealthPerspectives vol 15 pp 147ndash156 1976

[16] W M Thurlbeck and G E Angus ldquoGrowth and aging of thenormal human lungrdquo CHEST vol 67 no 2 pp S3ndashS6 1975

[17] H T JasonM R Bates andG I Charles ldquoRole of lung volumesanimal model of asthmardquo American Journal of Physiology vol297 no 3 pp L401ndashL410 2009

[18] R N Pinckard M Halonen and J D Palmer ldquoIntravascularaggregation and pulmonary sequestration of platelets duringIgE-induced systemic anaphylaxis in the rabbit abrogationof lethal anaphylactic shock by platelet depletionrdquo Journal ofImmunology vol 119 no 6 pp 2185ndash2193 1977

[19] H K Choi W E Finkbeiner and J H Widdicombe ldquoAcomparative study of mammalian tracheal mucous glandsrdquoJournal of Anatomy vol 197 no 3 pp 361ndash372 2000

[20] F L M Ricciardolo F Nijkamp V De Rose and G FolkertsldquoThe Guinea pig as an animal model for asthmardquo Current DrugTargets vol 9 no 6 pp 452ndash465 2008

[21] D H Eidelman S Bellofiore and J G Martin ldquoLate airwayresponses to antigen challenge in sensitized inbred ratsrdquo Amer-ican Review of Respiratory Disease vol 137 no 5 pp 1033ndash10371988

[22] G R Zosky and P D Sly ldquoAnimal models of asthmardquo Clinicaland Experimental Allergy vol 37 no 7 pp 973ndash988 2007

[23] E W Gelfand ldquoMice are a good model of human airwaydiseaserdquo American Journal of Respiratory and Critical CareMedicine vol 166 no 1 pp 5ndash6 2002

[24] M P Shampain B L Behrens G L Larsen and P M HensonldquoAn animal model of late pulmonary responses to AlternariachallengerdquoAmerican Review of Respiratory Disease vol 126 no3 pp 493ndash498 1982

[25] M Zabetian M Tahmoorespur and K Hosseini ldquoThe appli-cations of transgenic rabbits in agriculture and biomedicinerdquoJournal of Animal and Veterinary Advances vol 10 no 6 pp780ndash790 2011

[26] J LWrightMCosio andAChurg ldquoAnimalmodels of chronicobstructive pulmonary diseaserdquoAmerican Journal of Physiologyvol 295 no 1 pp L1ndashL15 2008

[27] A Churg and J L Wright ldquoBronchiolitis caused by occupa-tional and ambient atmospheric particlesrdquo Seminars in Respi-ratory and Critical Care Medicine vol 24 no 5 pp 577ndash5832003

[28] JWiddicombe ldquoThe airway vasculaturerdquo Experimental Physiol-ogy vol 78 no 4 pp 433ndash452 1993

[29] J Widdicombe ldquoThe tracheobronchial vasculature a possiblerole in asthmardquoMicrocirculation vol 3 no 2 pp 129ndash141 1996

[30] J F Murray ldquoThe structure and function of the lungrdquo Interna-tional Journal of Tuberculosis and LungDisease vol 14 no 4 pp391ndash396 2010

[31] A Hislop D C Muir M Jacobsen G Simon and L ReidldquoPostnatal growth and function of the pre-acinar airwaysrdquoThorax vol 27 no 3 pp 265ndash274 1972

[32] R G Crystal S H Randell J F Engelhardt J Voynow andM E Sunday ldquoAirway epithelial cells current concepts andchallengesrdquo Proceedings of the American Thoracic Society vol5 no 7 pp 772ndash777 2008

[33] M C Rose T J Nickola and J A Voynow ldquoAirway mucusobstruction mucin glycoproteins MUC gene regulation andgoblet cell hyperplasiardquo American Journal of Respiratory Celland Molecular Biology vol 25 no 5 pp 533ndash537 2001

[34] G J Douglas J F Price and C P Page ldquoA method for the long-term exposure of rabbits to environmental pollutant gasesrdquoEuropean Respiratory Journal vol 7 no 8 pp 1516ndash1526 1994


[35] E Hamelmann J Schwarze K Takeda et al ldquoNoninvasivemeasurement of airway responsiveness in allergic mice usingbarometric plethysmographyrdquo American Journal of Respiratoryand Critical Care Medicine vol 156 no 3 I pp 766ndash775 1997

[36] M L Conrad A O Yildirim S S Sonar et al ldquoComparisonof adjuvant and adjuvant-free murine experimental asthmamodelsrdquo Clinical and Experimental Allergy vol 39 no 8 pp1246ndash1254 2009

[37] F E Hargreave G Ryan and N C Thomson ldquoBronchialresponsiveness to histamine or methacholine in asthma mea-surement and clinical significancerdquo European Journal of Respi-ratory Diseases vol 63 no 121 pp 79ndash88 1982

[38] E F Juniper P A Frith and F E Hargreave ldquoAirwayresponsiveness to histamine and methacholine relationship tominimum treatment to control symptoms of asthmardquo Thoraxvol 36 no 8 pp 575ndash579 1981

[39] A El-Hashim B DrsquoAgostinoM GMatera and C Page ldquoChar-acterization of adenosine receptors involved in adenosine-induced bronchoconstriction in allergic rabbitsrdquo British Journalof Pharmacology vol 119 no 6 pp 1262ndash1268 1996

[40] P C M Obiefuna V K Batra A Nadeem P BorronC N Wilson and S Jamal Mustafa ldquoA novel A1 adeno-sine receptor antagonist L-97-1 [3-[2-(4-aminophenyl)-ethyl]-8-benzyl-7-2-ethyl-(2-hydroxy-ethyl)-amino]-ethyl -1-propyl-37-dihydro-purine-26-dione] reduces allergic responses tohouse dust mite in an allergic rabbit model of asthmardquo Journalof Pharmacology and Experimental Therapeutics vol 315 no 1pp 329ndash336 2005

[41] C M Herd D Donigi-Gale T S Shoupe D A Burroughs MYeadon and C P Page ldquoEffect of a 5-lipoxygenase inhibitor andleukotriene antagonist (PF 5901) on antigen-induced airwayresponses in neonatally immunized rabbitsrdquo British Journal ofPharmacology vol 112 no 1 pp 292ndash298 1994

[42] W R Marsh C G Irvin and K R Murphy ldquoIncreasesin airway reactivity to histamine and inflammatory cells inbronchoalveolar lavage after the late asthmatic response in ananimalmodelrdquoAmerican Review of Respiratory Disease vol 131no 6 pp 875ndash879 1985

[43] E M Minshall M M Riccio and C M Herd ldquoA novel animalmodel for investigating persistent airway hyperresponsivenessrdquoJournal of Pharmacological and Toxicological Methods vol 30no 4 pp 177ndash188 1993

[44] A J Coyle C P Page L Atkinson K Sjoerdsma C Touvayand W J Metzger ldquoModification of allergen-induced airwayobstruction and airway hyperresponsiveness in an allergic rab-bit model by the selective platelet-activating factor antagonistBN 52021rdquo Journal of Allergy and Clinical Immunology vol 84no 6 I pp 960ndash967 1989

[45] AWatanabe and H Hayashi ldquoAllergen-induced biphasic bron-choconstriction in ratsrdquo International Archives of Allergy andApplied Immunology vol 93 no 1 pp 26ndash34 1990

[46] G R Zosky P D Sly and D J Turner ldquoMouse modelsof asthmamdashwhat physiological evidence are they based onrdquoAllergy and Clinical Immunology International vol 18 no 2 pp76ndash79 2006

[47] J J De Bie M Kneepkens A D Kraneveld et al ldquoAbsence oflate airway response despite increased airway responsivenessand eosinophilia in a murine model of asthmardquo ExperimentalLung Research vol 26 no 7 pp 491ndash507 2000

[48] M Saetta and G Turato ldquoAirway pathology in asthmardquo Euro-pean Respiratory Journal Supplement vol 18 no 34 p 18S23S2001

[49] J Grommes and O Soehnlein ldquoContribution of neutrophils toacute lung injuryrdquoMolecular Medicine vol 17 no 3-4 pp 293ndash307 2011

[50] M Kinoshita S Ono and H Mochizuki ldquoNeutrophils mediateacute lung injury in rabbits role of neutrophil elastaserdquo Euro-pean Surgical Research vol 32 no 6 pp 337ndash346 2000

[51] H G Folkesson M A Matthay C A Hebert and V CBroaddus ldquoAcid aspiration-induced lung injury in rabbits ismediated by interleukin-8-dependent mechanismsrdquo Journal ofClinical Investigation vol 96 no 1 pp 107ndash116 1995

[52] R E C Altounyan ldquoReview of clinical activity and mode ofaction of sodium cromoglycaterdquo Clinical Allergy vol 10 pp481ndash489 1980

[53] N Gozzard A El-Hashim C M Herd et al ldquoEffect of theglucocorticosteroid budesonide and a novel phosphodiesterasetype 4 inhibitor CDP840 on antigen-induced airway responsesin neonatally immunised rabbitsrdquo British Journal of Pharmacol-ogy vol 118 no 5 pp 1201ndash1208 1996

[54] D R Ball Surgical Critical Care and Trauma Part 2 AirwayManagement in Trauma and Critical Care Scotland UK httpptolemylibraryutorontocasitesdefaultfilesreviews2012February20-20Airway20Managementpdf

[55] B Yahaya ldquoUnderstanding cellular mechanisms underlyingairway epithelial repair selecting the most appropriate animalmodelsrdquo The Scientific World Journal vol 2012 Article ID961684 12 pages 2012

[56] C Coraux R Hajj P Lesimple and E Puchelle ldquoIn vivomodelsof human airway epithelium repair and regenerationrdquo EuropeanRespiratory Review vol 14 no 97 pp 131ndash136 2005

[57] L M Crosby and C M Waters ldquoEpithelial repair mechanismsin the lungrdquo American Journal of Physiology vol 298 no 6 ppL715ndashL731 2010

[58] F Dupuit D Gaillard J Hinnrasky et al ldquoDifferentiated andfunctional human airway epithelium regeneration in trachealxenograftsrdquo American Journal of Physiology vol 278 no 1 ppL165ndashL176 2000

[59] B Yahaya A Baker P Tennant D J Smith G McLachlanand D D Collie ldquoAnalysis of airway epithelial regenerationand repair following endobronchial brush biopsy in sheeprdquoExperimental Lung Research vol 37 no 9 pp 519ndash535 2011

[60] J K Biehl and B Russell ldquoIntroduction to stem cell therapyrdquoJournal of Cardiovascular Nursing vol 24 no 2 pp 98ndash1032009

[61] C Mummery S I Wilmut A Stolpe and B J Roelen StemCells Scientific Facts and Fiction Elsevier New York NY USA2011

[62] A Alvarez F Unda M L Canavate and E Hilario ldquoStemcell and regenerative medicinerdquo Current Stem Cell Research andTherapy vol 4 no 4 pp 287ndash297 2009

[63] G M Roomans ldquoTissue engineering and the use of stemprogenitor cells for airway epithelium repairrdquo European Cells ampMaterials vol 19 pp 284ndash299 2010

[64] J E Boers A W Ambergen and F B J MThunnissen ldquoNum-ber and proliferation of basal and parabasal cells in normalhuman airway epitheliumrdquoAmerican Journal of Respiratory andCritical Care Medicine vol 157 no 6 pp 2000ndash2006 1998

[65] B R Stripp and S D Reynolds ldquoMaintenance and repair of thebronchiolar epitheliumrdquo Proceedings of the American ThoracicSociety vol 5 no 3 pp 328ndash333 2008

[66] B E Isakson R L Lubman G J Seedorf and S BoitanoldquoModulation of pulmonary alveolar type II cell phenotype and


communication by extracellular matrix and KGFrdquo AmericanJournal of Physiology vol 281 no 4 pp C1291ndashC1299 2001

[67] R A Wetsel D Wang and D G Calame ldquoTherapeutic poten-tial of lung epithelial progenitor cells derived from embryonicand induced pluripotent stem cellsrdquoAnnual Review ofMedicinevol 62 pp 95ndash105 2011

[68] E L Rawlins and B L M Hogan ldquoEpithelial stem cells of thelung privileged few or opportunities for manyrdquo Developmentvol 133 no 13 pp 2455ndash2465 2006

[69] D N Kotton B Y Ma W V Cardoso et al ldquoBone marrow-derived cells as progenitors of lung alveolar epitheliumrdquo Devel-opment vol 128 no 24 pp 5181ndash5188 2001

[70] D S Krause N D Theise M I Collector et al ldquoMulti-organmulti-lineage engraftment by a single bone marrow-derivedstem cellrdquo Cell vol 105 no 3 pp 369ndash377 2001

[71] N D Theise O Henegariu J Grove et al ldquoRadiation pneu-monitis in mice a severe injury model for pneumocyte engraft-ment frombonemarrowrdquoExperimentalHematology vol 30 no11 pp 1333ndash1338 2002

[72] B E Strauer and R Kornowski ldquoStem cell therapy in perspec-tiverdquo Circulation vol 107 no 7 pp 929ndash934 2003

[73] Y Jiang B N Jahagirdar R L Reinhardt et al ldquoPluripotencyof mesenchymal stem cells derived from adult marrowrdquoNaturevol 418 no 6893 pp 41ndash49 2002

[74] E Martin-Rendon D Sweeney F Lu J Girdlestone C Navar-rete and S M Watt ldquo5-Azacytidine-treated human mesenchy-mal stemprogenitor cells derived from umbilical cord cordblood and bone marrow do not generate cardiomyocytes invitro at high frequenciesrdquo Vox Sanguinis vol 95 no 2 pp 137ndash148 2008

[75] D J Prockop C A Gregory and J L Spees ldquoOne strategy forcell and gene therapy harnessing the power of adult stemcells torepair tissuesrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 100 no 1 pp 11917ndash119232003

[76] E Mansilla G H Marin F Sturla et al ldquoHuman mesenchymalstem cells are tolerized by mice and improve skin and spinalcord injuriesrdquo Transplantation Proceedings vol 37 no 1 pp292ndash294 2005

[77] J L Spees S D Olson J Ylostalo et al ldquoDifferentiation cellfusion andnuclear fusion during ex vivo repair of epitheliumbyhuman adult stem cells from bonemarrow stromardquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 100 no 5 pp 2397ndash2402 2003

[78] F P Barry and J M Murphy ldquoMesenchymal stem cells clinicalapplications and biological characterizationrdquo International Jour-nal of Biochemistry and Cell Biology vol 36 no 4 pp 568ndash5842004

[79] F Zhu G H Guo W Chen Y Peng J J Xing and N YWang ldquoEffect of bone marrow-derived mesenchymal stem cellstransplantation on the inflammatory response and lung injuryin rabbit with inhalation injuryrdquo Chinese Journal of Burns vol26 no 5 pp 360ndash365 2010

[80] S Aguilar C J Scotton K McNulty et al ldquoBone mar-row stem cells expressing keratinocyte growth factor via aninducible lentivirus protects against bleomycin-induced pul-monary fibrosisrdquo PLoS ONE vol 4 no 11 Article ID e80132009

[81] N Gupta X Su B Popov W L Jae V Serikov and M AMatthay ldquoIntrapulmonary delivery of bone marrow-derivedmesenchymal stem cells improves survival and attenuates

endotoxin-induced acute lung injury in micerdquo Journal ofImmunology vol 179 no 3 pp 1855ndash1863 2007

[82] L A Ortiz F Gambelli C McBride et al ldquoMesenchymal stemcell engraftment in lung is enhanced in response to bleomycinexposure and ameliorates its fibrotic effectsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 100 no 14 pp 8407ndash8411 2003

[83] M Rojas J Xu C R Woods et al ldquoBone marrow-derivedmesenchymal stem cells in repair of the injured lungrdquoAmericanJournal of Respiratory Cell and Molecular Biology vol 33 no 2pp 145ndash152 2005

[84] H Yuhgetsu Y Ohno N Funaguchi et al ldquoBeneficial effectsof autologous bone marrow mononuclear cell transplantationagainst elastase-induced emphysema in rabbitsrdquo ExperimentalLung Research vol 32 no 9 pp 413ndash426 2006

[85] B E Sumpio J Timothy Riley and A Dardik ldquoCells in focusendothelial cellrdquo International Journal of Biochemistry and CellBiology vol 34 no 12 pp 1508ndash1512 2002

[86] T Asahara and A Kawamoto ldquoEndothelial progenitor cells forpostnatal vasculogenesisrdquo American Journal of Physiology vol287 no 3 pp C572ndashC579 2004

[87] K Strehlow N Werner J Berweiler et al ldquoEstrogen increasesbone marrow-derived endothelial progenitor cell productionand diminishes neointima formationrdquo Circulation vol 107 no24 pp 3059ndash3065 2003

[88] C F Lam Y C Liu J K Hsu et al ldquoAutologous transplantationof endothelial progenitor cells attenuates acute lung injury inrabbitsrdquo Anesthesiology vol 108 no 3 pp 392ndash401 2008

[89] T He L A Smith S Harrington K A Nath N M Capliceand Z S Katusic ldquoTransplantation of circulating endothelialprogenitor cells restores endothelial function of denuded rabbitcarotid arteriesrdquo Stroke vol 35 no 10 pp 2378ndash2384 2004

[90] D Y Kim J Pyun JW Choi et al ldquoTissue-engineered allografttracheal cartilage using fibrinhyaluronan composite gel and itsin vivo implantationrdquo Laryngoscope vol 120 no 1 pp 30ndash382010

[91] Y Inayama G E R Hook A R Brody et al ldquoThe differenti-ation potential of tracheal basal cellsrdquo Laboratory Investigationvol 58 no 6 pp 706ndash717 1988

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Direct factors

Indirect factors

Infiltration recruitment of

inflammatory cells and formation of

granulation tissue (extracellular matrix

fibroblasts endothelial cells neutrophils and

leukocytes)

Acute lung injury acute respiratory distress syndrome

Lung injury

∙ Lung infection∙ Aspiration of

stomach contentinto lung∙ Inhalation of

highly toxic gases

∙ Sepsis∙Multiple trauma

with shock∙ Large volume

blood replacement

Figure 1 Direct and indirect factors that contribute to the onset of acute lung disease especially acute lung injury and acute respiratorydistress syndrome

the attacks occur Common clinical conditions associatedwith the onset of ARDS are pneumonia aspiration of gastriccontents pulmonary contusion fat emboli near drowninginhalation injury and reperfusion pulmonary oedema aftertransplantation or pulmonary embolectomy [5] Workers inchemical producing factories can also develop ALIARDSdue to inhalation of noxious fumes

Other than harsh chemical agents and biological factorphysical force on airway lumen also can induce hyperrespon-siveness In a study done by Latahir and Yahaya [6] they useda novel brushing technique via tracheal perturbation usingan endotracheal tube to induce injury on the upper airwayThis technique applied amount of forces on the wall of thetrachea which caused bleeding in the area of injury Later onduring the recovery period the recruitment of neutrophilsinto the area of injury will lead to the onset of acute lunginjury This novel technique can be used as an alternativemethod to induce acute lung injury on airway lumen withoutdoing minor surgery which requires skill

Asthma chronic obstructive lung disease (COPD)chronic bronchitis emphysema bronchiectasis and cysticfibrosis are the primary examples of the pathological con-sequences of chronic lung disease Smoke inhalation is themain aetiological cause but occupational environment andunderlying genetic makeup also play significant roles indeveloping chronic lung disease These diseases are usuallyassociated with development of airway inflammation airwayhyperresponsiveness (AHR) and mucus overproductionCOPD refers to conditions characterised by chronic orrecurrent obstruction of air flow including chronic bronchitisand emphysema Chronic bronchitis results from recurrentepisodes of acute bronchitis or persistent and noninfec-tive irritation of bronchial mucosa This disease is usuallyassociated with emphysema a condition characterised bypersistent dilatation of air spaces and destruction of theirwalls [7] Asthma is characterised by a variety of features

including reversible airway obstruction airway inflamma-tion and increased airway responsiveness to both physicaland chemical stimuli [8]

2 Rationale for Using Animal Models toStudy Lung Disease

Lung diseases are extremely prevalent worldwide thus thedevelopment of prevention strategies and new treatmentmethods to reduce the worldwide burden and increasepersonal quality of life is crucial As for any other diseaseclinical investigation and epidemiological studies are neededto advance knowledge and improve disease management [9]A biological model is needed to study the mechanisms thatunderlie the development of lung disease at both cellular andmolecular levels

Human models of ALI have been reported in severalcase studies Human models were used in a clinical trial todevelop and test a novel therapeutic agent targeted to cureALIARDS [10] However there are drawbacks to use humansas research subjects Humans can suffer from other diseaseduring the experimental period and there are risks associatedwith experimental procedures (eg inducing a low level ofinflammatory response) Therefore healthy volunteers mustbe thoroughly educated about the potential risk of participat-ing in a given study Various ethical and pathological issuesalso limit the use of humans as an in vivomodel

Thus animals are preferable as an experimental modelfor modelling the human respiratory system They providean experimental setting that allows researchers to study theinteraction between the immune system and a functioningrespiratory system Choosing an ideal animal model for usein lung disease studies must follow several criteria The mostimportant is the ability of the model to reproduce the princi-pal aspect of human lung disease The model also should be









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Journal of

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The Rabbit as a Model for Studying Lung Disease and Stem Cell