Diaphragm structure and function in emphysematous hamsters

WO REIO, RK WILTON. Diaphragm structure and function in emphysematous hamsters. Can Respir J 1994; l(l ):53-58. OnJECTIVF.: To dete rmine if muscle fihre injury. reduced force outpul and fatig ue of the diaphragm accompanied hyperinflation induced by experi mental e mphysema. DESIGN: ConLrollcd and ra ndo rni1.ed . ANIMALS: Adull male golden Syrian hamsters: seven control and seven experime ntal em physema hamsters were ~mt.lied . INTERVF,NTIO:\'S: Following anesthesia. experimental emphysema hamste rs received a translracheal injectio n of elastase. They also received injections of P-aminopropionitri le every other day ro r five weeks . After five weeks. all h am~ters we re anesthetized , and thei r diaphrag m and lullgs ,w rL· excised . MAIN RFSULTS: Me asurements llf" the excised lungs ~howed an inc reased residual volume. functional re~idual capacity and total lung capacity in experime ntal e mphy, ema hamsters. In vitro physiolog ical study of diaphragm strips showed no di fference in force output but a greater fa1 iguahility during re petitive stimulat ion for 5 mins in experimental e mphysema hamsters than in control ham,ters. Histo logical examination did not show significant muscle injury in the diaphragm of experimental emphysema hamste rs . CONCLUSION: T he greater fat iguability and absence of muscle injury in the cliaphr.:igm o f emphysematous hamsters may rc~ult from hyperinfla tio n decreasing diaphragmatic load. Alte rnatively, muscle damage not detcclablc at the light microscopic level may have contributed to increa~ed fatiguahility of the diaphragm of experimental e mphyse matous hamsters.

An animal model of increased resistive loading by tracheal banding hamsters was developed and marked diaphragm damage and infl ammation were fo und (3).Another animal model, experimental emphysema, leads to profound hyperinflation and fla ttening of the diaphrag m (4) but li llle change in pulmonary resistance (5).In experimental emphysema, the ventilatory loads imposed on the diaphragm may he greater because it is forced to pump more frequently in a less functional position.Alternatively, a less functio nal position of the diaphragm may result in decreased recruitment of this muscle and greater reliance on other inspiratory muscles for ventilation.The purpose of this study was to determ ine if muscle fibre injury, reduced force output and fatig ue of the diaphragm accompanied hyperinflation induced by experimental emphyse ma.The effects of this type of loading on the diaphragm were assessed by exa mining: fi rst, muscle morphology using light microscopy; and second.in vitro phys iologica l function of costal diaphragm strips.

Animals and groups
Fourteen adult male golden Syrian hamsters were obtained from Charles Ri ver and cared for in accordance with the principles outli ned in Care fi'1r txperimenral Ani111als: A Guide for Canada (C anadi an Council on Animal Care 1980 and 1984 ).Two groups of animals ( 16 to 19 weeks old) were studied: seven control (C) and seven experi mental emphysema (E) hamsters.Thei r mean body weight was 138±20 g .

Experimental protocol
The experimental protocol received ethical approval fro m the University of Britis h Columbia A nimal Care Comm ittee.Follow ing premedication with atropine and diazepam, hamsters were anesthet ized by intraperitonea l inj ection of sodium pentobarbital (6.5 mg/100 g body wei ght).A small incision was made over the trachea, and the tissues overlying the trachea were bluntly dissected in order to vi ew the trachea.The animal was placed on a 45° angle and 25 units of porcine pancreatic el astase (Sigma, Missouri) per 100 g body weight diluted in 0. 3 mL of saline so lution was injected Lra nstrache;:illy into the lungs of the E group.A similar amount of saline was injected transtrachea lly into the lungs of the C group.Hamsters recovered from the anesthesi a. lrn mediatl'ly afte r the lranstrachca l injections of clastase or sal ine 1mly, each an imal was rotated side to side to fac ilitate distribution of solutions throughout the an ima l's lungs.
All hamsters were monitored dai ly and E hamsters received intraperitoneal injections o f ~-am inopropionitrile monofumurate (Sigma) ( l mg/g body weight) , a lathy roge n that interferes with el astin synthesis ( 6) , e very other day during the next fi ve weeks .Four-and-a-half weeks after the trans tracheal injection o f e lastase, fo ur E hamsters had the ir left carotid artery du-onically cannulated (3).Five weeks after the transtracheal injection of elastase, hamste rs \\-Crl' aga in anesthetized with an intraperitoneal injection or sodium pentobarbital.Under deep anesthesia, the great vessels were transected and the diaphragm was excised with th1: ldt anteri or rib cage left attached.A stri p of muscle from the left anterior costal diaphragm was excised to determine in vitro physiological fu ncti on.Biopsies from the ri ght costal diaphragm were cut and quick froze n in isopentane coo led to the temperature of liqu id nitrogen for histochem ical anal ysis.Frozen ti ss ue was stored at -70°C until process ing.Lung vo lumes were de termi ned fro m the excised lungs .
Lung volumes: Just before sacri!icing the hamster by tra nsectio n o f the great vessels, the trac hea was ti ed off at fu nctional resid ual capac ity (FRC ).After removal of the musc le biopsies, the lungs and med iastinum were removed.FRC was determined by vo lume disp lacement of the lungs in saline .To determine residua l vo lume (RY ). the tie on the trachea was removed.the lungs were defl ated to at least -20 cmH20.and the lung volu me was de term ined by volume displacement in sa line .Lastly, total lung capacity (TLC) was determined by formalin fixatio n at 30 crnH20.The weight o f the med iastinum and lung • was subtracted from the volumes determined.
In vitro physiological function: Under a dissec ti ng microscope, a 2 mm wi de strip of the cos ta I diaphragm was exc ised wi th its attachments to the central tendon and a small segment of the rib cage.The muscle was quickly placed into ajacketeu musc le bath containing Kreb' s solution (115 mM sodium chloride, 25 mM sod ium bicarbonate, 1.2 111M sod ium phosphate monobasic, 5.0 mM potassium chloride .3.4 mM calcium chloride, 1.2 mM magnes ium sulphate and 2 g D glucose/L) held at 37° C and bubbled with 95~ oxygen and 5% carbon dioxide .T he muscle strip was mounted horizontally bet ween a motor arm using a small sta in less steel cl ip attached to the central te ndon and a force u•a nsducer (±50 g, Kulite) using a silk suture tied around the rib segment.T he strips of muscle were electricall y sti mulated via platinu m plate e lectrodes.Optimal length (Lo) was de fined as the length at which maximal twitch tens ion (0.2 ms duration) developed.All subsequent contracti ons we re perfo rmed at Lo. Isometric tension output during variable frequencies ( I, 20, 30, 50 and 100 Hz) were determined.The diaphragmatic muscle strip, held at Lo, was then exposed to a fa tig ue run of repetitive bursts of e lectrical stimu li (duty cycle: 0.20, 30 Hz, repetiti on.: 24/min) for 5 mins.The percentage force loss during the fatigue run was ca lculated.Next, the muscle was blotted dry, the rib cage and central tendon were removed, the muscle strip was weighed.and the cross-sectional area was determined using the tech nique by Close et al (7).Force output was expressed per cross-sectional area (specific force).

Histological analysis
Cross-sections of costal biopsies were cut IO µm thick on a cryostat-microto me (Reichart-Jung, New York) and stained with hematoxylin and eosin.The area fractions (AA) (8) of normal muscle, abnormal and inflamed muscle, and connecti ve ti ssue were determined from cross-sections of each biopsy.AA were determined using a light microscope equipped wi th a camera lucida (Lahophot, Nikon, Japan) and a computer program for point counting that was developed in this laboratory.T he image of a 50-point grid from the computer monitor was projected via the camera lucida onto the fie ld of view of the diaphragm cross-sections viewed at 400x.T he points projected on the c ross-section were then ass igned to one of the following categories: I. normal muscle; 2, abnormal and in fla med muscle including necrotic muscle.viable muscle with abnormal morphology, necrotic muscle wi th intlammatory cells. in fl ammatory cells (where no outline of a muscle cell was ev ident) and effu sion : 3. connective ti ssue -fat or collagen ; 4. no count -if the point fell onto clear space, nerve or vesse ls .The number ol" points in each of thl: first three categories was divided by the total number of poi nts in these three categories to determine A.I\.  ---------------------Figure 3) Plot of specific force over a r(ll1ge of.,•ti111ulatio11.fi•cq11P11cies.Afean ± standard de1•iation.C Con trol; E Experi111ental cmphv-se111a (50 and I 00 Hz) stimulation: and fo urth .fati guability (force drop during repetitive stimul ation).If the mul ll varialc analy sis indicated a significant di fference in a grou p of related parameters between the C and E hamsters, Student's t tests were performed on individu al parameters to look for spec ific differences between C and E hamsters.

DISCUSSION
E111physe mat ous hamster, dc rnon,tratcd marked hyperinflation assoc iated w ith increased in vit ro fati guahili ty o r the diaphragm.In contrast to a model of incrcased rcsi'>t ive Joa<.l ing (3).we found 110 significant evide nce of d iaphragm muscle inj ury and in1la111111 at io11 in emphyse matous hamster,.
The profound hype rinlbt ion in emphyse111atou, ham,11.:rsl\:sul!, in marked 1lallcning of the hamster di aphragm a, , hliwn o n x-ray (4).T hc diaphragms in the cmphyscmatou, ham, tcrs of this study nmy have been placed in a similar tl attencJ, k s, functional position.This may ha ve resulted in <. leereascd recruit ment of the diaphragm and greater rel iance on other prima ry and acces,ory inspiratory muscle, for irn,piral ion.Alternati ve ly. the nll:chani cal di saJ vantagc of 1he diaphragm in a more flallcned position may have forced thi, muscle to work harder and possi bly rc~ultcd in rnu,ck inj ury not dctect;1blc by o ur techniques at the light microscopic lc\'cl.Either decrca-,cd u,c of the di aphragm or undetected J;1111agc.such ;1, Ji sorgani 1ation of the myofibrillar structure (3).rnuld expl ain the incrca,ed fot iguahilit y of the di 1phr;1gm in our sludy.A lhird pos,ihility is that decreased elastin.due to ad mini strat ion or P-aminopropionil rilc. in the various connective ti ssue layer, su1Tut111ding individu;d rnu,cle fi l)I\:, and 111yof"ibrils re, ultcd in inneascd fotigua hili ty o f the CllStal :,.trips from the E ha111stcrs.I Iowcver.there is li tt le in l' o rmati o11 in the lite rature to su pport or refute thi ~ hypothesis.
Our results differ from those of others regard ing the in vitro fatiguability of costal diaphragm strips from E hamsters.However, very different stimulation regimens were used in previous studies.so it is ve1y difficult to compare the results.Both Farkas et al (9) and Lewis el al ( l 0) found that costal strips from E hamsters were less fatiguablc than those fro m C hamsters.The protocols in both of the previous studies were more rigorous than our protocol, using a higher duty cycle (0.:13 to 0.:18 versus 0.20 in our study) and a higher repetition rate (60 to 90 repetitions/min versus 24 repetitions/min in our study).It is quite possible that different processes such as high energy phosphate depletion and proton load contrihuled to fatigue in the more rigorous prntocols used previously, whereas the glycogen depletion or ionic imbalance may have contributed to fatigue in our less stressful protocol.Little work has been done to investigate mechanisms contributing to fatigue in different stimulation paradigms used for in vitro fatigue protocols.We selected to use the stimulation protocol previously outlined by Paga la et al ( l l) because it appeared to be the least stressful protocol relative to others in the literature.
Anothe r factor that may have cuntributed to the differing fatiguability resul ts hetween previous studies (9, l 0) and our study w as duration for which experimental emphysema was imposed.In previous reports by Farkas et al (9) and Lewis et al ( l 0). the duration of emphysema was approximately six months, compared with five weeks in our study.The longer duration of emphysema in previous studies would have provided a much longer time period for completion of any adaptive process in the diaphragm.We chose to study the diaphragm after shorter duration of e mphysema for that very reason.Hype rinllation had been pre viously reported al'ler five weeks of inducing emphysema (5).If di aphragm injury occurred, however, it was less likely that reparative processes would be complete relative to a six-month period of emphysema.
There was no loss of force output at low or high frequencies of stimulation in E hamsters.Specific force is related to muscle fibre type composition ( 12) and the cross-sectional area composed of viable muscle.It is unlikely that the fibre type composition of the diaphragm was changed by hyperinflation induced over the five-week period that emphysema was produced in this study.because three previous studies examining the effects of much longer periods of experimental emphysema on the hamster diaphragm found no diffe rences in fibre type proportions (9,10,13 ).Regarding cross-sectional area of viable muscle.we did not find a significant increase in AA of muscle fibre damage although abnormalities were seen in the diaphragm of some hamsters.
The lack of change in force output in our stud y is similar to results from two early studies that showed a similar specific tension of costal diaphragm strips from control and emphysematous hamsters stimulated in vitro ( 14.15).In contrast, a more recent study by Le wis et al ( I 0) found that the specific force of the diaphragm decreased in emphysematous hamsters .In all of the previous studies ( I 0. 14, 15), emphysema was induced for longer (six to 18 months) than the Can Respir J Vol 1 No 1 Spring 1994 Diaphragm in emphysematous hamsters five-wee k period in our study before diaphragm function was studied.Emphysema was induced in seven-to nine-week-old animals for six months in the study by Lewis et al ( 10). and in 40-week-old animals for 23 weeks in the study hy Farkas and Roussos ( 14 ).Supinski and Kelsen ( 15) did not describe the age of their animals, hut emphysema was induced for l 8 months before study of the diaphragm.The younger animals used in the study by Lewis et al ( I 0) may have enabled a greater transformation of muscle characteristics including myosin isozymes during the animals' development and.hence, a difference in specific force output.
We did not find any significant differe nces in the proportions of normal muscle, abnormal muscle and connective tissue in the cross-sectional area of the costal diaphragm.Other investigators have found that, although experimental emphysema produces profound hyperin flation, there is lilllc change in pulmonary resistance in hamsters (5) in contrast to the 6 .5-foldincreased pulmonary resistance in the six-day tracheal ba nding model ( 16).The overall work or breathing imposed on the diaphragm is likely much less in the emphysematous mode l relative to the six-day tracheal banded model because of hyperinflation llauening the d iaphragm and the small change in pulmonary resistance.In addition.due to the time course of experimental emphysema, the work of breathing wou ld change much mon:' .gradually than that or tracheal banding.Thus, the workload imposed may have been insufficient and/or too gradual lo cause any consistent amount of diaph ragm injury.although muscle damage was ohserved in some animals.It is possihle, howeve r, that excessive loading was imposed on the other inspiralory muscles leading to overuse injury; however, we did not examjne other inspiratory muscles in this study.
In conclusion, hyperinflation resulting from experimental emphysema in hamsters does not induce significant muscle injury in the diaphragm shown at the light microscopic level.Increased fatiguability of the diaphragm in emphysematous hamsters may result from hyperinflation decreasing diaphragmatic load.Alternatively, muscle damage not detectable al the light microscopic level may have contributed lo increased fatiguability of the diaphragm of E hamsters.