neurotensin akira tange1

15 Pages·2015·6.21 MB·English

Biomedical Research 4 (1) 9-24, 1983
DISTRIBUTION OF PEPTIDE-CONTAINING ENDOCRINE
CELLS AND NEURONS IN THE GASTROINTESTINAL TRACT
OF THE DOG: IMMUNOHISTOCHEMICAL STUDIES USING
ANTISERA TO SOMATOSTATIN, SUBSTANCE P, VASOACTIVE
INTESTINAL POLYPEPTIDE, MET-ENKEPHALIN, AND
NEUROTENSIN
AKIRA TANGE1
SecondDepartment ofSurgery, Kyoto Prefectural University ofMedicine, Kyoto 602, Japan
ABSTRACT
The distribution of peptide-containing endocrine cells and neurons in the stomach,
duodenum, jejunum, ileum, and colon of the dog was studied with the peroxidase-
antiperoxidase technique using antisera to somatostatin (SOM), substance P (SP),
vasoactive intestinal polypeptide(VIP), Met-enkephalin (M-ENK), and neurotensin
(NT). 1) Somatostatin-containing endocrine cells were distributed throughout all
parts of the gastrointestinal tract. Met-enkephalin-containing endocrine cells were
located only in the pylorus and duodenum. Neurotensin-containing endocrine cells
were observed in the duodenum, jejunum, ileum and colon. Neither substance P
nor VIP immunoreactivity was detected in gut endocrine cells. 2) Somatostatin,
substance P, VIP, and Met-enkephalin immunoreactivity was observed also in neu-
ronal elements throughoutthe gastrointestinal tract, whereas neurotensin immunore-
activity was detected only in the small intestine. Immunoreactivity to all peptides
exceptneurotensinwasfound innervefibers and in ganglioncellslocated inboth the
myenteric and submucous plexus. No neurotensin-containing neurons were seen
in any layer of the gut wall. Somatostatin-positive nerve fibers were distributed in
the myenteric plexus, circular muscle, submucous plexus, and lamina propria mu-
cosae. A large number ofsubstance P- and VIP—positive nerve fibers were seenin all
layers of the gut wall; they formed a dense network especially in the nerve plexus
and lamina propria. Met-enkephalin-positive nerve fibers were also demonstrated
in all layers and formed a dense network in the myenteric and submucous plexus.
Neurotensin-positive nerve fibers were found in the longitudinal muscle, myenteric
plexus, and circular muscle, but only a few were seen in the submucous plexus.
One ofthe most excitingfindings in recent years arealso foundin thebrain(7). In thegut, these
has been that many of the hormonal peptides peptides, which have been termed brain-gut
initially isolated in the brain are also present in peptides (7), are located both in endocrine cells
the gut, and that several gut hormonal peptides and intramural and extramural neurons.
The peptides identified in gutendocrine cells
includesomatostatin (SOM) (1, 3, 21, 25, 26, 32,
1Presentaddress: FirstDepartmentofSurgery,Shiga
34, 44,46), substance P(SP)(38,41, 53), vasoac-
University of Medical Science, Seta Tsukinowacho,
tive intestinal polypeptide (VIP) (9, 10, 43),
Otsu, Shiga 520-21, Japan
10 A. TANGE
enkephalin (ENK) (2, 24), and neurotensin (NT) distal colon) werequicklyremoved. Thesewere
(16, 20, 25, 40, 45, 52). dissected into small blocks (5-10mm in size).
There are also ten or more distinct types of Care was taken to avoid any damage to the
enteric intramural neurons which have been mucosal and muscularlayers ofthesespecimens.
distinguished byelectrophysiological, pharmaco- They were then postfixed for 4811 at 4°C in a
logical, histochemical, and biochemical studies fixative containing 4% paraformaldehyde and
(for review see 19). Certain peptides reveal 0.2% picric acid buffered with PB. The fixed
different functions in the enteric neuron system. specimens werewashed with PB containing 15%
Immunohistochemicalstudieshavedemonstrated sucrose for 48 11 at 4°C.
that gut nerves may contain SOM, SP, VIP, Cryostat sections 20#111 thick were processed
ENK, gastrin/cholecystokinin, bombesin, NT forimmunohistochemistryessentiallyasreported
andotherpeptides(19, 23, 48, 54). Mostofthese by Kimura er al. (29), using the unlabeled anti-
peptidergic neurons are intrinsic to the gut (48), body peroxidase-antiperoxidase complex (PAP)
but morphological and functional analysis is developed by Sternberger (50). Free-floating
complicated by the fact that the same peptides sections werepretreatedwith0.3%Triton X-100
are also present in extrinsic neurons of, for for4 days and treated with0.1 % normal bovine
example, the vagus nerve (23). serum for 1 11, and incubated with specific
It is worthwhile to study the distribution of antisera raised in rabbits against somatostatin,
bothendocrinecellsandneuronsofthegutin the substance P, vasoactive intestinal polypeptide,
same tissue preparation. A systematical analy- Met-enkephalin, or neurotensin for 5 days.
sis of the distribution of peptide-containing They were then incubated with anti-rabbit goat
neurons has been reported using rodents (48). IgG (Miles; .1: 200) for 2 h, and with PAP
Thecanineguthas been favored in thestudies of (Dako; 1 : 200) for 2hat room temperature.
the functional roles of the brain-gut peptides The characteristics of the antisera used are
(4-6, 30, 31, 36, 37,49). Althoughmanystudies shown in Table 1. Each antiserum was diluted
on the morphological analysis of peptide-_con- with PBS containing 0.3% Triton X-100.
taining elements in the gut of this species have Specimens werewashed in PBS containing0.3%
beenreported(3, 10, 16, 20, 24~26, 30, 40,4.1,43, Triton X-100 before they were transferred from
44, 46, 52), a systematic investigation of the one serum to another. In order to inhibit the
peptides in both neuronal andendocrinesystems activityofendogenousperoxidase, tissuesections
through. the entire gastrointestinal tract is were treated with0.1% H202 and subsequently
lacking. with 0.1% phenylhydrazine buffered with PBS
In the present. study a detailed description of afterincubationwiththepeptidespecificantisera
the qualitative and semiquantitative distribution (51). Finally, each antigen was rendered visible
ofseveral bioactivepeptides, i'.e., SOM, SP, VIP, by reaction with a solution containing 0.02%
methionine-enkephalin (M-ENK), andNTinthe diaminobenzidine and 0.015% H202 for 10min
gastrointestinal tract of the dog is given. The at room temperature. Stained sections were
present results in the dog support the previous mounted on gelatin-coated glass slides, air dried,
physiological and pharmacological findings on and washed with running tap water. These
the distribution of these peptides in the dog. sections were dehydrated in a graded series of
ethanol, cleared in xylene and covered with
Entellan.
MATERIALS AND METHODS
Control tests for the immunostaining were
Eleven dogs of both sexes weighing 5-8 kg were done by replacing a peptide-specific antiserum
used. They were deeply anesthetized with sodi- with a non-immunized rabbit serum, or by
um pentobarbital (Nembutal) and perfused incubating with each peptide-specific antiserum
through the left ventricle with 1-21 of ice-cold which had been absorbed by an excess amount
phosphate buffer saline (PBS; 0.1 M phosphate ofeach antigen (10-100 jug/ml).
buffer, 0.9%Na.Cl, pH 7.4) followed by 3-41 of The peptides used for the control tests were:
an ice-cold fixative containing 4% paraform- Synthetic somatostatin, substance P, Met-en-
aldehyde, 0.3% glutaraldehyde and 0.2% kephalin, and neurotensin obtained fromProtein
picric acid in 0.1M sodium phosphate buffer Research Foundation, Osaka, Japan; synthetic
(PB; pH 7.4). After perfusion fixation, pieces VIP was generously supplied by Professor N.
of the stomach (fundus, corpus, antrum and Yanaihara,LaboratoryofBioorganicChemistry,
pylorus), thesmallintestine(duodenum,jejunum ShizuokaCollegeofPharmacy, Shizuoka,Japan.
and ileum), and thelargeintestine(proximal and
ENDOCRINE CELLS AND PEPTIDERGIC NEURONS 11
Table 1 Clza.='acreiv'stfc:s ofAntisem
H-__A—n--t--i-s----e---ra to ndgilutlion Source
Somatostatin OAL 272 1 : 8,000 Otsuka Assay Laboratories
Tokushirna, Japan
Substance P 24410 1 : 5,000 Immuno Nuclear Corporation
Stilwater, MN 55082 U.S.A.
VIP R 502 I : 5,000 Professor N. Yanaihara
Shizuoka College ofPharmacy
Shizuoka, Japan
Met-Enkephalin 41310 1 2 4,000 lmmuno Nuclear Corporation
Stilwater, MN 55082 U.S.A.
Neurotensin R 3511 1 : 4,000 Professor N. Yanaihara
Shizuoka College ofPharmacy
Shizuoka. Japan
RESULTS the mucosa. They were characteristically elon-
gate in shape, with short processes which often
Endocrine cells containing somatostatin (SOM), formed terminal knobs(Fig. lb). Intheantrum
Met-enkephalin(M-ENK),andneurotensin(NT) and pylorus, thecellsweremainlylocated within
were distributed in various regions ofthe canine themiddlethird ofthemucosa (Fig. 1c), andhad
gut, butnoendocrinecellscontainedsubstanceP a triangularoran ovoid shape,withathickcyto-
(SP) or vasoactive intestinal polypeptide (VIP). plasmic process commonly reaching the lumen
On theotherhand, neuronalelementscontaining (Fig. 1d).
SOM, SP, VIP, M-ENK, andNT were all found The SOM positive endocrine cells were also
in the gut. These peptides were present in located among the cells of the intestinal glands
neuronal somata and fibers, with the exception below the crypts in the small intestine (Fig. 2).
ofNT, which was found only in neuronal fibers. But the majority were located in the lower
All thesepeptidescould beobservedwithout any portions ofthe intestinal glands, in pyramidal or
pharmacological pretreatment with vinblastine bottle-like shapes (Fig. 2). In the colon, they
or colchicine. were scattered across the colonic glands below
The distribution of the different peptide- the crypts, in a shape similar to those found in
containing endocrine cells and neurons varied theupperdigestivetract. Aroughmeasurement
according to the portion of the gastrointestinal of the number of SOM-containing endocrine
tractandthelayerofthegutwall. This distribu- cells indicated that their population density
tion is diagrammatically summarized in Dia- decreased towards the anal side.
gram 1. As the distribution patterns of each Neuronal elemems Many intensely stained
neuronal peptide were mostly uniform through- SOM immunoreactive nerve cells and varicose
out thedigestivetract, thepattern in thejejunum and non-varicose fibers were found in the
is shown as a representative case. The submu- myenteric plexus of the jejunum (Fig. 3). The
cousplexusinthecaninestomachissosparseand cytoplasm of the SOM positive neurons was
difficult to examine systematically that it was densely packed with imrnunoreactive granules,
not examined in this study. thenucleibeingdevoidofreactionproducts(Fig.
3). Long nerve fibers running in the myenteric
plexus appeared to connect each ganglion. A
Somatostarfn Immzmoreactivity few immunoreactive fibers were observed in a.
Endocrine cells SOM immunoreactive endo- discrete region near the deep muscular plexus in
crine cells occurred in the entire region from the the circular muscle of the jejunum (Fig. 4),
stomach to the distal part of the colon (Figs. whereasnoSOM positivenervefiberswerefound
1,a—dand 2). In thebodyofthestomach, these in the longitudinal muscle (Fig. 3). Numerous
cells were scattered among the exocrine cells of SOM-containing neuronal somata and fibers
the gastric glands (Fig. 1, a and b), and were were present in the ganglia of the jejunal sub-
predominantly located within the lower half of mucous plexuses(Fig. 5). There were no fibers
12 A. TANGE
UB US
ENNDOECRCELL LTNMOUUANSCDGLLE MPYELNETXEURCS CMUSCLERCULARSMPULCEOXUUSS MCMUULACFO-ZSSA LNPAAROAMPR
STOMACH
DUODENUM
SOM JEJUNUM
ILEUM
COLON
STOMACH
ouooenum
SP ueuuwum Ii‘-|
ILEUM)in-1-
conoml-I
STOMACH
l
4;
DUODENUM
_
Vlp JEJUNUM
ILEUM
q--.-
COLON
STOMACH
DUODENUM
.._L
M-ENK JEJUNUM
ILEUM
COLON 2
I SIC sroMAcH I
ll.
DUODENUM
NT JEJUNUM
--l1i_
ILEUM
COLON L J W
5Very high to high density of imrnunoreactivity.
High to moderate density of immunoreactivity.
Moderate to low density of imrnunoreactivity.
Low to very low density of irnmunoreactivity.
No irnmunoreactivity observed.
‘Mix Not examined
Diagram 1 Summarized representation ofthe density ofSOM-, SP-, VIP-, M-ENK- and NT-immunoreac
tive endocrinecells and nerve fibers in the dog gastrointestinal tract
ENDOCRINE CELLS AND PEPTIDERGICNEURONS 13
.
. -.-
:_.,».>>_-,-.;=-.,,2._»-;;,_---.---: .-::'-,.:11;I-<../:,<;-’,;':-.,-.=-.'-,>-~.-»,;-..;-==-,:--',.---.\.'-'-;---;"-:.-;t.1.I-_..;_.=E-.;--,..'==1>;-15,<._-,[email protected].'.>-=:'¢!€,z;-.~=?..4-;1-;;:r-g;;.._t;;==._-'--..=_.2,/'.3-.-.'<z\-‘g"-r;-:-’,,.>,<:1'-_-r:..;;-c.-.-...--_:j=-;.=.-~:-__¢.,..‘.>-;..-:,;.._---.,-,.1.,.=.>_‘"-,--.“.;._=\-f;:'.-;~i-.e',/-=';:/--’»>r-:_-*i-f1-'...-.---:.'.§.{1a.;,n:;--..5:,.;.-=-;,'=-4'f_z-;,:-2//»~?-_¢,--__e=~r.--r“>--25>5-1,--5=.5--_3--;_-‘=..1==_./.'-;;:.;a=-.-;-'-;..=.---'..:-.---;-_-_..-.__.-"=-_-_'_.---_=_;:';_'>;'..-,-=3.-:,.>17;.,;.',=-,='<=<-.-.,,=,~-r,s;,:--5,?.:__."_‘:_...:-;..5;;;/.-":.I;;.:-_'_ -"- --
f'<1.'..i;,~<$-_.;_--. I =/./,.,»>c,;;;;z.-;s-.-.;";_.2-;e;»;~-";¥fn'a='_-.-;='._=;>,4;:;/r,v3:;-.-'.-_--'.-.-=_-';s-;-_ r/,=-;;-"=-.=,<-;:=:_=_=-_"-_-;;-;1=-_=--:-;;_t=;=_:-:-1;-14'-3-.»;-. -.:§_=_.'==;".=-:-;-;~. -
¢_-,>.;-._,-=-=_;f.-2,_._.:I_-.__:_==_!2.--_:jg--.-.Q.'--.,;-':;-;:-..9:_:--;2;.,;-"-.I-_-1_.F_._-._-;._::_-~_;,,_>;_-.-:-i;_>_;-;'-;._;;-..:.,-_:’,.»_--_r_,;_:_!__,,..-_=i=I=z;>_2,"-:,5.:;:-:__e,>a~,-m_-:',,-;--_.-.-,-;-_-..";~=-'-'.-:-1.: __-2_I_'_-.--3-__:;.---2?.._--. "Q-F.
<1:ii §-:-=-.-.=:1- s- _- ".1:-1-;-::;"::-:3"-zr .-r.an-:;:§:-;-.;.;',.;;_-:-'.=5;;;I;-@;¢:;"-:-:;a-=-I--" ..-;.-;-; -.=.-.1-_-:.- -
-_-4..;_-.<=-_§.;:;__.=-::.;{=?=j,;'.;;-5a_;3;'-;_1.-'--._\.;‘,=:;j.§{,‘§;<.,g-,-<,;-.;_I:§_-'-..:_-§f':_;<'..;?._'.-;;a{in,--2;-.a_-.;_:-1_>'£-;;,-'=:_:.'=.,-_.=5;;-r.-_--.i;;;;:;;;,__;-;-§;;-_-;_.;ra;;-.;.a-'.=i=- -,'.rf>,.P__;,.;{_;1:i;;;;;.;;2-;-@}#5-};/g{-»E,¢~{.-;>;;;.;-:;_-_<:___;_.;,;;._-_:-,;_-;.-§,/2;%¢.§;;p.-~_.--;';>;;_,-ja@,_1.,:,-.5;1.3-;2,=23§9._'-=,.;-,f-,_;..-.;=£;_;-;,--';,-__1;__:3,=-,_-'.;-;_.g-;-.;;_'._-=;%j-:'.--_,=:.-_,-;'a=_v'-__.;_.-;,=-__.j-,¥-_;.;.__g:;,x3~,§;,>j.5:-._;;},-,-,:,-;-2§,;_,;_3-2%..,4,;-_.;_j.--5-..-5;.--',"5--;_.-.-_:; . '.3
..-2..'=I_-=-_:--"..'_I-_.'_;:;-P_2=,,;*-gJ;P-;;.=-,;'_--_’--._"-_:-;>:i,=r\,-{,._f=;‘=2.,§,>,€,1.-.§-~.-.~;¢‘&!\-2“;_<,-¢.;r'}<'a-t,{E15-1;:'::._'.:'\'.=|‘::.-_1-_=_i._-;..-_=4,(1->,'-(4_»{;j_}41"'#1.i.§',¢-f-5I.11\*:‘§>-a"=,-_n‘7-_/-.-*=5-.iii-’-t"35_r2_3_-:2="--,+»i;1-"1t3’§§...<';>;j‘=»é--,i_"~ii='-l%,.-g--_$.';.s-ei;i*b=?k~k“QSv..-X--g:§,4,g-..<,-,fi>_,.-f-_Kg,».,<“1g;,;,_,g»,_,-/n/,,_",-e,-An.i-,,t,r/;_,£=;,\;__.-;.,5=,g,-;_1._{_,[-.533;-..--.;._:-.-5.-,=._-_-_-;..-...-';-;_.=_-_-j-_-_;;-;_-:5;§-.;-_g.:;;_.=__-_-:;...-._.-;.=__-',_2--.;_--__-;__
‘/56 --- /“\- )'§)f1)$>g-J ,,,... /,_-/,,-;_‘:u,,,<$;,-;§* ,,-.,.,;,/. »/4, ...}:3’4.. . :" I.
,-;=:’>e -;0='-.;-'-';,;.:.,'>:-;-.'.--."-.:-1 '_\,.\,3*-_=2I.-.-;.=_\?,=.-5--;='.-§1:5;=2;5-1,;;?§='{-;-,>=-11.. .-vi.,,_;f-f=_'.'=.’,§'<.;\,\.§;:5-5'' ,-:. .e_-’-,'.;;:;j-.2;-.1-5='/;?_=.'-I;.-;1.=,'=-._;;;.;.-._';_';§-;;;~-;f;5J_--,,-&,1?;';.=?-.;''5:-’-*1_-”‘.'.-.'."' ?;_- ;._--.--3_-
..:I;_-_-;..=:_'-=-.:-I_,.I=,;_;.-=,.=-,-:..-=.,=:=,_.;..,_=.-;,._-:1;',:__1-:.__,:.2-.-'-,_.-',..--_=j.-;._,v-,§’--i1§1:.--''5...»_--.,2.:-.'1;''._.-;;-._-j-.:,.;---;.<;.-.-':;..=..-;=:.;-_'.>-._%;'._;-;-;>;..'_.-;.;..-;§:.;.,n,§.;-;.;,-._¢-'..:,4.,=5.::.--='5--.-4'5--2..';.-.;_I.;...¢..;.-,l=-'.._;;».;..£',;1._,,_-,i,,L,.»j.f.:»Q;,.¢.-.,-§,;_=_..-:,_.~-§i.,;-_;.:;:',.t___"-;,“,17,;=,..,;-.,;,x;:.=2.;r,-Z»'-r.}:'?--,-:=>-=N_--':_§=;,=,,-:.Z--;'-§:j_=_-,-;>=,;,,.=;-.<.s;;—;;:_:.-,=;-=E,:-,.2-_,--:,;-:.;-r,==,=;=1_==,,-..='.,=;..=!_-:I-.=,.-:="',>u..'.s..'-‘,1.=.5=>_‘,_-'_".,<=.,§\_.:-:'.Ia'==;/.“£.;,';i_ft<-'-'-,._£$.~5’.-;.'>'..;;=;,:..-_-F7.=..=._:'=--=_;.-_-;-I..5=;..g:,->5!._-;$--..6-'.3.\;--:{_>,»,=.>;i;_"1;;-,':,;-_--_;=»_>...-.-,.-=:~;\=_:-,;..=;'=,.-,;'.3-;1‘.;>,=;=;;j;;:,;<_--,,=;/;=-_,-\:;.,,..-;,-;,-¢-;:;,.;-';;.-;',_.2_-=i:i-5;,;;;;----2;-;-3:¢.-:/,;,,%g-,g=>.;:;::-?::-,;-;-..<-;§{-_Q5=’22-,;,.2;‘;;53'-,‘;5';_-i3=--2;j;=-:,i.»z_=-=f-.?'@x:_'---2'.=_,;--;'Ig:ij;..,;_'_»='-'_:£_.;'>;1--.j!-a._.=i'g,,_-g_=;,,_=-._-_,<'_--2:_/5=;»-4;-,<,-.,‘_,-_.-"._-__-_-_,-I-_,;.-;';,--_I}-__:.;... -:
'.._==ii€'.§==- I .;?=’<:5>p==-'_.-;-'.;§''1I?,.K $'4,?-'-T--,4,-' .--;?=.!:»;:e§:I;,g{{;:.5:._=.' ;-,= = ._ .'
-__--1_-_"-.>_"-9;=4;i’<\'t¢j;'’;-,'~.=>'6./1’-----_..-".->,<-»‘'.'$§!<E1,-.;,.-,{/I_.<-._T;..==_f--=..;.';,;‘--E.i'.f:‘.-.I‘_3’.};§.'J;I§_-/_5_/v=»"-",j-3;.'/<-,_’..._ -;-2,. ._/>-1--’2~.,;'@_/.i;*;;>=4,-.1'.£/'j-<e’..>3¢,»-;'§-,=‘§=*;='§;-:'_l_:;'.;,__'-.,_'.I__._'_,3-,~.._,'--,-'-Q}‘._-'.;;;'-ztgii:-1-;-::5,1?-’'-_‘-,.V .-./, _;j-'i_<§;';a ,~_‘.i',=,-2-'i5'.T=.'f-':I1.'-3.':=§‘.=§;:%;;j;=-?;';.i-*.=--;.-‘'--:==
,'.--"_"_":.',-,-._:1-=.j:-,--,<:;-=.._*-,’1-_; .__.,,- .¢‘__ . Q.-»,.''._'-_-,--.,;,:>.=-.5,,_-_,..\\'.2,=-'=-_-..'-__-.'-_=£{§;é>§-.__...-,__-_,;...¢<2,;/_;n'r=i,i,\-iZ\1\:1”-,,,,‘,5;“.;£-,~1r.:r,-.'’,§.¢.>:_?-._._:-'-?__.,'-;_.';.-'-___.-';.,'-;_,='.<-_;-,;,§,‘,-,_gA=-_,1.r-H,{<...21_-;-;2.__-1--'.§>_/=2.-§;g;,,;,;._I,;_._\_2I-5.,5.:»>';,1¥,.5,$_-.~;;<.-,a->re.-.,.,--2,_;.-._,'-).r_.,_;-. 1-'-_-';1.;.-j.3;;>___!;_.-;..,;,-_./-,_-K.=;'.--'-=..1.-. j-.
3.;-=',;-_= -_;-!-===-/,'.‘-'\,-.---1‘-."-3:" -----'.=a>:.-/4."-=.1--:1-;=':-air?-ff.,./'1'.-.-1.-'>'’§.<r-;'/;~1'-,-',r=1.==:9,'-.';-ii:-= '--=‘-. --~~---:-A ~->4?-':--="-./,':1-:-'- -=.-:1-.-;-=-.- :- '
§1-;-r---xQ5' ;-'.5;-_-.<;5'~.-'>'£:‘.x=e;;=:,-:-_'-.- .>-;‘7J.-I>;-'-i:I-f;F3'?-'.'-‘.5,-gifFf1;-I;'.;';§-.';=';:>I7.’-.'.'-_2-'.-;-f.-3'-'2;Mfg;-_r5'5-I{';'£’,}:f-§¢. =3.'-.--.'--;.-';--1:-5:»;-{-i->?:;y»';'3»;-:§-.£~ .'-_'----_- -,.§f:'-:-;-'--.--_.=I {
G~ --.=-;-_-.;-.-_..-,:;;-;',=;.--:2>5;-6.§-,;M--.'._-I,-,_...£;_-:--=r,:-.,1..-._:.,~“-_~:;<.----.;2~;._I;-".:;.;__'-§'>=-.—-.._;';-;:2.-;g_--.<3---:-2_.;-=4...;1-5_.2=-;.--,.51-/._,-==;r..,;.;'-=;j_:='=!f-_if-_--1.;‘.,'f»-.5:j=5'I--:3,5;'ii-F.L_;--./.5-;;._:'>-_,=I--:§';\5¢<._=-_§_>>-./_51Z-jg5A1;'2r.=-;;§.-.:-;'-.j-=.'.-!-t.':..-'r-.;.1i.ij;,>§e,-r;>;;,j:-;.<3--_1---n;..-,r.-;_..-_...--.~».--__=_.<.---._-:_-.I;--j;;:.i;;;f-=-_f.,-z=-<.;}_-,r9.--.;-._;----..-;-'..-;.;-"-r;=.:.r-I§_;-.=_--.._--f,;..=-i:'_.-4---1.=;1;.;.¢r.<:>i‘?<(;/i.-§_->-':.;;;..';--j./=,-.H-5-.-*.:_''>§-'?,>-.;§.-..-.-.=-:{._l~=:4';==g1-?.-"'/§}5-/fs:5I-;-y§-z<;'_e;»--'-r»-‘>-_.;5<--3..;-=-_j_-5-.--"I.t;I._-;-.-'1;...;<‘t-_-_-.=-:‘-'-*-.,'=-<='->--'>»;..;-.'7;<:-@-;1./=>-.;'J-_.:=-.=-_.--,‘.-.r.e_.,,;?-;--=,,-_<;-;.,¢-.i-;3>._v',Z¢s‘§i_1‘,i-;I{-isJ..._--.--------—-;.;-.----------.-'--‘.I1--.-;;.-,_';,t:-"--.-..‘+_I.1-,-j-,.-__-:__I
is ..
._-..-.<.-'~.§.-»2-~.,2-.;;;..;,..»_.1.-'-.1:;2‘.-:-1/~/.---,M.I. .,¢_--,,--',-,-,....-,..-._.-_-¢1,.--;_;'-"»-‘_i_A.'_/,§,_e;j.».>>A:5r’',.;-(,_,=_-..---,-=_-_-,_..,4;,_.=:_‘.'£V.j1_-..I._1-;--.-’2..,2v.;’1=;->25A_;.;’5,3-,»*-'3-j5;-,-:$_2f._",,/.-_‘=¢_2.;_-.,1;..__-__--3::._1_-.¢=_;'-_‘._:f-=_'.f--I,-2.1,;-z-..-1'43‘-I:= ,_r»_e.5.-"‘/>--/‘”yNjy».-¥-*‘-<,\.;',2-,-‘€.<-:.:2...X.<.,,<;~¢2-Z.1¢-_,7.¢>,-,,.:;;-1',.2=.;;,,2¢:;;;,-$"-."_-._.,.,W,')-;.--i2,,(~é;2,‘~.z..-.;,_,-._¢'_--..; Z
~q.-..y»-»sl-"--~"-.=-1-’<-'>’.<--..V»9:1“-'3¥,"2'/5:;._¢{.-?'/-.;5',<=f;;1<g':/;.r'f./»T;;"-3;.;.<>.=.’.;.--'.;.:;4§;.g1ja,;r-;;--‘.;.1.11--.,;-.r.a;,ayé-._;";==._':1_;?,/~<.:=,y_.g>.;/y;.,;>_;>;-;.\3';>§r_;<?1;P.I=§,.5;-*.3'',_.e‘-''--.,I.*5x-:“m»Eg2iQt*,-;=;ig1.r5i-;sz;;.,.;:z;=;;-.:e.:r'-:-.;3;=t'.:;=-;.;;e%;¢;i;€3€1';;=;‘‘;._--;I.=,;,.==;..f=;!,§<=;!,:,<¢>--~9rf-="ai<Z/>’--1';'~'=.-f{_}:-".::-i‘f:f'_-/ /':,5ei/-./;-<-".-'s".m-l4,-/2€/"-,9,,;3/;.-2:=;_;.-~;_'-'-=g*>../.;={">-1'<W'l=»s...,'¥¢-;1<"2:;.v..‘>;1."Wf4f.(_2‘/':.,e“rt">/3->‘>;/"e‘~=r.;.<>-4-7"'5/--¢I“7-'
is-:E.1,=ix2-!-;."-f'\--_1;1:--2-§i.,1‘.»’;'5?:.-Z-.:_...-.<-—.-:=2,-i“i_sr-).-’-"-"!5;-,;.”€>r;:'-*..’(../ni;2--_>{1-_-‘'..f<.‘.’.-f:;-".;»-"-_/,s-’¢'.rr-.-“—--‘~i,?-rr-.-’;_,=.-.<':=:_:""a.-_;;y"5--'"<>is'5:'»£_¢_Z:,>r’'.--3,:1-f-i_=§-=--.n,.<'-.-::;'"',J=-.'._~(.\--E=-?*~;61>'-:/>‘a.E$’:./_=i'.q_%-</5‘''?».,.,-'=;/=‘",?;.|>:¢,-="E-..i>:</.>,~;,.1._.r"‘-.:5'X_.-'"_.2.;.;»>>:'>,;:;‘,\.~.,?1r<,_-/‘\I3=§,"-_.'-.e»‘5,‘;-5’">\-r,.g.-i=§»%2-yII’<$~.»*-,'-,;"_’}?j?-§’§%.=_7"4._:~;,“~'P,-y‘>_‘:.»25-K1/;~5.’:.;>."-"g’",4~:_/’-.--¢,_i->»:~.-.*-._E*1._i:-~‘-,)‘i.”..,‘‘§*--,.*/.<@l».§;.>/*,»-.r’si.=='---'W/gei/’>s-gi._“1aH1;;..v»4r’..>,sI-*';;r<-as_._-§-..2/-/>\flF(9-Q.-’;.5,-;~-~a/,-..}.-..-.24"95;2f1_>..»;~?5<,,(g3£:.<r§~t-,>..<i~----:;‘0,-‘3a-§9>2a-:,_.r‘."=..’:<...f§:E'45r-,,.--=-.1¢?-X..s.,#,,i¢.~>»_‘*’'-='>i:.r»_>v\._'-,->.,<-\=:.._-.F._=,s-»-{''',:-r.=Q';s*~éL-.-'t,,,’-=-?’‘.-§.,5..-:.'»..:;+€»t?-r“'M.,51-._:5A%.-4M»-g-*,,a_*.‘s\‘r’.3”=.5.,_i\i’-./<'_-4r-;_t/>/1I-,Z1,--?s-:<‘-c/§;4>-s<'y‘S\-tJ.,1.r,_-1°’.3Ai.;1/’'x./'. ‘--"-."'f,'.i\__f‘._W<,.'1--I—\'*5"‘I»,.*'''.1".'-',—.',@;-.3—__i,,.5.-_--'f~e'-"----.?=<-.-.7-—_’.."Z_=:¢-2"r_.‘~:%w";-~"!»’<'("?~”KI__t‘e1.--.1.r.J~‘-'’.-.1‘-1_-_'\-\-.',.:'>.-“,~1.K---'-F'2’----.-;.81.,\\:'\.-2~.>-“3.‘:-_=“-‘/-Y-<-:''?_“;1''‘5:--.~§.':>-;‘-.:";-?;-<'->-’'’.;-’-“:P.5~"=-I,-'':.-.".,=».4;T»{,K---:.._,<,€&5'':1;~-..1'-:»3;':.5-§’_’--T.--;»=i~>I.--.;j1~g-».'.-,X5_-g'--’.f$—’>/=~;~-—=_:;’;,4-=:‘:“,=<=§~";'-§-'—-,.-::9,.-1:=W>~-=:§..~.',J-"-’.l"/.-_5=.'Ij,---.’.a,”.;-\'./i.=-'»'I--,".®-.,?;_4t-:"2=W5.fv1.';..:‘_:.;';-w--'.";-<.T>=i,1:=-~’>..._.5;'f-_:E:-.::-~»-,:,=."2.--=e§»-2’;1-".,1.¢5,/-=~:-/-"-’’1:,I.<'/z.._~I;>'i-/=/.¢-,».="E,;3_§m2/I-"1*.fii,;>--.*-~s‘.;=~».~;“'_>//Ii.*:-2=»-1-2'I,,51.»;»..-—'..’2..":-.4<.9",t>;..4.===-t-*-:=-)c‘~.<){.‘=_v?-,/,"s"».:<K-’.._S-;Z.;.;S).-'‘~.-_,=-'uq-'?,.;<.v1>=m=-AEn."_'‘.ti¢,-,*t*>1,Qis4-Q\.-,»i;>=.'""2g;<2">Q..'s%;3'1‘8‘.,;=->4gF:e?92‘.>,.":-»‘,-~--3!2'-,(»,"-'"a\t./=_g-";“4~..r-v-Y_.‘.-}—.;_-),:1.~£,._=.\_..,§.2-'_.w_,'‘.\<._.\2=-‘-\*=.‘-\_‘.J£<-,/.i>".”:;-w-Y-.¢§2.-¢{1»,;t?if,./f.1,“_4:»-»:,;:%’j-“-,-.1¢/>i»-,; -Y",r3»§r/f1bI:f;*-~"i->‘.,?~“‘-1._5’_-?,.5i,"”.l,,m~Wa-._>--.f}{';t~r=."."rl-a»,‘f;2.2_~.‘-f6"=_f'é)w1.e:ev-“=-Qd.»-~;I-s1ei/ e--.eJ‘}.»1»‘W&;Zv-1;;;T1?/a:1l,1'‘r-.'"3»“1’.-,=¢/_—’%-3’-,;-E.‘.”"-;1/£-\j:e¢-.#‘1<-i.3“t§".’_'’g1v-:-‘>:-;:-;“a>.-.4~-i“i,-,>.*9>it.;‘‘Fee*‘.‘Qa-4"i.,,»<“a~_,,M.2I{f§e>5.2.UV_A5-;-',i.1,%»"¢"“-5w§32*.,J<-’L4.;/.,".¢'-».€é&_-2:_,'-'1I"/x;-s,;-r,s-'=.i-r.—_.2:)fr-,,!y-z5:,,;;>.,‘15¢*:¢.;fi"'11,,6~.//;1‘.’.,.;,;.-’).,(»-,-(-7;./,**<34m/<"~€<9=,xr-{”42‘;>_-a_1_""''i51-§i"§-;w"X.?g";1:i-/-.47_§;Hvr’<?m{:'.,_.1-~-.-<".i’1:“_?--,,z'-5-"2:;»5'¢;,<.-'»w§<\,-->-;'-.‘.,:''.<*8”--,P<..._-->-,a..%:'r>>'1,/.;.:-5:;<.§.r~;.;<F_;'‘-;r9,.,4<=.<a».-a<_5"',,>...:45?~%-~,<~ir>’...I.§_1,:.-'>:;r~~8-.=;‘r;.<;<3=-g.-s‘.‘n_»<t~-*';1§»,‘s='z:re2:/,\“£;./2%-~¢g#.;~.:2>-~,=--.»‘-1=-»',.=:<=.I§'»;'§<=,22e3-L~,-~@,‘=_:=,_=';»<">%-a-=i._-~:%--.--""-1:-$=:.....i1?~’¢~3_,>'-ii;:;:Ts.2-_r-t,.gj/#-2<,¢--_F*‘5=‘<s.-'g.re...>:;t‘=..-@¢;2_;"‘g»;i--.-;=.{-<i‘s3<.’»-"-,<‘<t?>'-»..-.i-.-.g/<5.---;'E<¢,;..-=_--.>-§,~,:Wj=€<>-,--5,§<-,..--,4.:<.-’i2-,»3*4Y4>~gt£'_.=.-~<1,==;s3-3”*’.e:F'..§i»,‘;s.‘@¢_,---ia-("‘*=/iJ3-.:’.i.,2.;5:.~‘-,'.’3-(...,;_/.=-:€i¢_*13.--2.='?.-:‘.;“‘3=';:--vf=i9';%-<i,":,._.r-;.,-.~./fs€?//.,>.i;»:41»,2<5§;%5?:,i,<.s{.<¢,4--.~?t.,e..'>--!-"‘.x-.,”,;,.§.3--§,~-<r,»"4'5,2,\-*.:1<.;\»':_./-s=,_~4~=."-~,..‘i-;-.:,2-.:=-”§=é-<");-~.1’.--ifi¢<;"'''_r>,’~<.;>-n-'.,*’,',,r~5?*.;=.::»~.~»/"s:§.;.">_-,i'=t':é<J.-,;":A.ni’.-»/’$>"s,'_-fi§.-.'p.?1‘.,-e.-:Iu*.".;--t§‘4-g-".~->-§--;5~>,.rr.g.;p;_.’'fi“~j=?v--2’4.§a€.*r1‘-<-;g§I;:52s.5.;.*6g-:'-->_/-10-“.,\,.5R'-..r'~‘I52-\1g“'(&'"“*Iuv“:‘§1.‘6r'..,\\-2-;‘R§.-\e=,i_¢.i-».‘§=2.}?;”_-Jfi,_»/*'-Z,-,ip‘Q?.-e,.,f,2,g'=‘»/sr.-’J5Z.i_.§/i.¢*»’s-5-ss,»-‘,rim2_9'€{,»-;c43'\=,=x{.2?i‘_--§1s’rt.:3ir-e<'/,i;;-i,»1‘--.;-Aizt.”g-,e.»Q\-.:E_»i,¢--?';»;;<'r"QJiK*:.",s-.‘:,”/-sW5\-.2-_»§'e,_w..$;';."...'»/:,1.”e._:,,¢--~x"f(:/»e-.’a-,...-=}é*‘.1';.>,73n,“.>~4,.--'t=-.\_,',/-»"_:,r<,-5,-»-’’:--*F.;:.-.-,-9-7=,:_.¢‘L=-“e1~-—2./'’-1-’-...2;2>’,>’.~,;--.‘;;r-_.---,.1-/¢.-,<1-=-,/~i;3--;8“=..,1_.-=.Q,4--~__~2'€,2-_-'-._-:-.,»$=-'/.‘=-i?~2-/-'.’ir,2.‘i1--‘3-='--*:---.=~..>>’"=222>Jg,.-~>f-.t3;,,,i-’-1-.’-_-i;’-><>\_:tfE"-s:§/-.i./.‘>-»..>:..w”<-n-!.-,~'z-.";==.'\w_-._-,.;.:.-_i-::i_>;..T~JQ._,,~’''e.,_=>.__A?~.‘f.\.>i<-.§_§‘2.o._>-}i:-.i~>i.’t’,/I'-1Q,.-*;<,e--<,’'>ya>S‘£.n,;§1'‘»(-r'lK.>$+>_;>2»#f?,Y.ir.‘..';,e>>>r--'-‘'~-»_-_nK,4fyrA'%"'<t>g',‘if/r;=_._‘'-5-.'/"-?,'-;;:=s>-1,..r/a-7-",,/.f-..-.’/a.".,sP'i1-'-;<-‘4,)e¢>Vr._i~’","’§v:-,J/<>GE*§w,--'.v4’-i-.5.-_-<:,.".»-i1-;$..?i.--~,7’2’>'‘!>=,.‘.g)---3;5-“>/“.-.-';.w?n.;=<5:»4»i‘.-Bt-‘,l?<,¥’:»:;=..‘&4~§_:.~,~,-J-£,§~-:_=5-"==:.¢9>;;.-=/i;---iiJ.:-1r.,.--y\>»+.*.!.4'.="-5>.-»--'<-5..'--’_r-=s3.,r.¢_2¢.'i<.-i‘'<r=3.=;,=2;"_.>'-?;;_-"-;-E=,,53~-..'/_-..':'I.,’J,r=.r1’r:=,.-:,>:.:._/'=,;\_;.>1:5.2-2\-~.~-.1"r';-".=-.;’._:,.>_=>-€:/-.~:»"-/..z_=,:'-_~"=’~-->.;—:.>225;-T..§-,_~,;;,<=/>$,::>;;6".-"--§==»>»5:;_;.:4.»;?..-?1-.=:-'-1..-’12-f'§:,.’=-->--’i"=‘.?¢.R-:".y-§.51‘.5-h.;‘IiINi/‘!"’
F_<"=%§’:4-;--r.?-“"’">‘="' ;__r§5__§=§-.'fi 1,-L<.e§£.F"I“f/I-14¢.‘-';'*;*_,€'$-.-__-x-1,.- ”'Z-‘Eff/1%.-Q‘.',1~?s*‘>’---'“.i-;.§.:;‘,¢'I» >~
>/.<=-'2: ~ . .-.__4..;.\-=.--;.--,_.., - - . =,__.,q__ §.>-___¢g.-.-'- _==>__.,.. ._~- ‘ 7 '>,_ '_ ,,-- > ga5.-.
. ... W./~- ....mi4’%...--. -.--1;-,-,.__=-...-:11.:_:;.. .- *4‘I.2 ,-¢.-"--e-fir.fi--gJ,_?,,».-,525_,/_.,.‘:a___t_.¢;-,.=......_,;-<i-x'__-_-__,,.-__H_.m,,"-=rr*'ie£.-.=.r3'§/e>~r=;<‘;:t'-g*T»§<_-"’*f?/-?"/.~?"5é-aJ1-"e.:'_»€+>=Z-r;:.2/5-4r$-at
W, _ 2;rFw-¢ 's~'5§’§.3.»1°...'/.;,.<sj,_.._,-.n, ,.' 7‘Y--5'= xi-.- -,:/,,>7>_.2;-i..»:,,»>-.;-<",,==~»,,;¢»i,,,»;%-- -.
--'=-,--’.;g.,,,->»1>;-.1F".=:§.}j__...~;~5'.d.2£/%=1;,i'-/?-->..‘'??\’_*'’'._-.-_,,?>»’e,"?<»‘?a2-;"3-<».§s.m<,i_>,-/yM,;,.;a’;.-rx"*‘?i‘‘.>§'.=ir’-:>2ii.§i.'*“=>..-.’.1.1'I.g»°3/..”‘.S‘l?»,.,":.»"'_"i..f"-"-='”2_".”__‘“_a-’%I3g"-g-‘..'é’’j-.."'2-f=l'"~-\-._I9.t--."_t"‘".g-"-"-al‘€ti:‘“ré_.‘~“.a'i‘-r'(e.t1tti-z.-_I'-<>3-,-x.-»xrQ\-1\.?/."'F2’i;"-_“:EQf’§i_-;-_‘:;-/r7*2‘.4"//‘_§-f.’i/<a."'rFr2-l,,",>,.,,-,,2,._*<-.9.,»7=,|ej_A'‘,'$.)N.,)t\jZ\\";£&.,,'v,.,_.'i'Z_,5‘2§§"\gI§.}?.'§§;‘._.<%§.:?ia<'p,‘=‘;é"g'{fi;'_,:_'.£~<r..'M-;''_;._?_.‘:,‘_;"i’§i=:-‘.=f->¢-.;I-§
-;2a]1=>'>'1n;.:-_>";'‘2r>-e;-‘;1>/‘<-;~/<.::'2/-<1»,»-§=>-"‘-..)=i_',5.<;,=».,;‘-1s=,-,,-e":'é,-.\->=>»§--,'F;;):..-..<=i"@»,_¢.t4;’/,=/F>=/:<<=_;,'e.==,&:-r,s*-,2-,'"'.§">-.~-5>%-=,~>2.3:-4.:-,_,->:<»>£¢/$-1’.-\;i.3(,--.>r\4_..v;-_f,-._‘y)=»;;<%,1.\---;-,$’,‘3;.4»\r?z{§-W§1=’’,:-¢--3x--,-.‘“”/-.-,/.i<.€*§i\ -x5;../¢-§*.»..:i.>‘l-''-W-i>,f<;''\5->\-.;';-;;w§.>-,;/-~4-..;:z,t_''i@\,_2\*?,f.e=.\’-.§=//>./.~-;§>:./-§;:"£=,,,0,;P<-='-,~*=:’¢r_-/.>;:;;.'i>*'§rr»,',P;?.-42*.w.gv<§=:a‘:<?,/”>,,5-.;-=i;:9=,>=~.>=,>--;:-x-2-:i,'.;>>>1,-¢;»-,-’;--5.x~§=1-/?4_p-/..,-.-.7.€\,-.§"-.-;':--.5’\:>-r*v;.'-£;.<1%ik’e2_...$=‘;-»-:'''i>,§<;f-_r/,.',-'2;.49(<.,;.-y:..e-49..?-'1.-'-./'-,>'=-»-\,-5',£i-?->»>->4'’;,/><,42_s'_-:q44=:-<.-2i‘3.>;'=je§.;I4-e;=>_.;<-"?=‘'r=<:>3i§;;.$-r>,.--'.*\..=/¢»:,/é."?..i;:>=’,r='=4‘,y.5(=..',=-X,»1>.-41\’;.1-:,'..-'1-,-'4-.-=--»;.9/-_-;-;>'"_,-,,-~?’§'“..1:-'-’}.i-)e_-91I='-%-'1-.'$¢'4v,,_~'-J:-..~W-:<--,9-:e;:\1,»i-_,/s-.%-.-1''I--;_ff\/i-»-,Q~-f,§-4:'\-9,1,i..%,.'-\;.§,1:-L,‘,-;“$,§-‘-3X_"4X;--.\5P“,2’3,i.-2i»r~\\"Q- -,,9<:?“3>.3‘:9)‘,»!$,y1v-2-‘./._,fl''~..<;5-i.-:f/~"fj’4».fi,-A/iE-’=y»?E$-.,r./_I{?’._j_=:/>-/.2,2/\1:-;~'=--e;';f--r<§'.§’;fi}-i<j’i..¢-f9~Y~<”>/'/}:_¢,=;-_‘-=.-Z-.,",..£*;;»{/3-\r/i,'<-9>»-_/.¢r5"‘Qj¢§3iig&*I-//,-:£;/,-(.’J3”‘3-<5',1¢5;.=.;5%‘°'-.»’?--~'r':i.-’}‘f,!i*/)1=‘<~€»''"»:»;.''2.5;='7_,;-?‘§g",3§--_:5¢r’l§i-”4.,-i_e5i_?‘‘:’’!..=_*i'-r-x".'-?-';i,*wi£-g->"-,§r-'-z,,.3i.,%§,/¢3-;2,:<-;';/r-=\{»'/ef'4-=2f5_';3:.--‘}=-;€§..’-.;,.:"g7'$<-,,->-z_-:33-;f<.>°X;.<-',2-,.~»?f-?3gR»é3»f-.-:w*i<e;r¢'2‘>'fl¢fi'rr*/\§r-)>j3.!”-,i‘'.-€t:»<‘_‘>-i1‘£,/;</r's:Q';§6)-*§h2?.;-v.-_.’=’2-§37§~.4!?/?1>‘r",/{_‘'J2£,>fi;fi5'2,E§‘,,Pr1jf§.:§<r’.'»=/_?,51v.r4‘;.(§?§*‘>-4;/-;)£‘-“\4§i‘'.%:>-5-,=§->)!;E-$'1‘-\,’3?./~.=;'v;-9i5/I;r0',;>}53.r->:;1};::.n>-};;.w-%;i;--tt!>£»§-2x?“.-,.é:>,:-i1?$,-;9°,rJ1?2?{'_I;'-rf$.§=>7+-Jgrr(iW.r/i-;;i-9,d-J,Ir<?r/a9;a1'2e_§4si-g§;:aa--§1i-=!,x(»:i-.w'n<P.';:{-4)-.I..>(i;;r:’-=f‘r,9i.-4;':=F5i‘€~a;,-i’a.5;}/1:-4~~9<»Iri-_'1-E-»../.2-r.:-1=;4-<=-'é.1,3.,*-2."1;':';3,-e‘;:/J-'.i;5§2---E:_1Q>i/n4\1;";7§{;-»=;;‘:-;5.;.%>}:.-5.01»:£1'-b?.:,;::.%,§'y-;i=-z_i:;.1/i’<£.';=~i'-§'4=§-fi??>"'1':;i-.{3.=>:f-=-:%-;..--5=?5e{<1,~-,.i1,%-4:;;§<<--;-,-;K5<.'.__:<.5r5>w51\14-z.';':5;55fi5..-/.='%y~4Z-£:\',5\1=;-i'/'/;4}/i;:=-£;t’,nfi-;j.'.:,;-?'I;3:";.-ge;65,-';-‘--.;'9}.fi'"-z.-3;:.-5.2'-K-5'v;e\Z1-'f.,=;-e9f‘;2'.-,,r?r?-5l<r;,,r,:!..=-i.>;/>.),-'.i'-.--'\éi>.."§.-;-f;'1"r._#3Qr',i-=>-=-!.;,;€"F(I;--}41‘<-_f')~;>.3’.:fr-_/-=,>',<=fo-'=,;i;,,3V.,¢'':'<::-}.;'3-$g,i:?1j§9,=;i},/;-:.5‘2-.i=,-i'<g.'=\';-.£:{/...;2’<>g/{‘f>-:T.-,:.='.§=1;;4;>_\§.=;.i._<€;-93i-_;9z.1>.=,:=:};,e,;'-%‘:-’\,I-::=>»-_>-.-&:._:1a,f-'-::.r.?-;$_-,§s,....1F-§,=-;<_:",>_--5._-;-.i='::i;._.f;3.1-,:31,)_;-:-3,i§L7:';-=i;<;”;""-.:>i-,-,,-_';--;-3§-;!~=,.;-*?.,---:~f,;r=.‘-';;z--=§;;§=‘'=--::=;,n--Z.~.>:2'-=.--_1i-‘'§.--1.-=.¥--ai<-.E2=.'=;.=--'=:..;-£4..§,2_-;I>._',-;::<;-f-»';¥;r._?=>*=;,=,,,,-,*;=_\-1;‘-,.I‘-=§§<--,_>=-;,<,Z',:"..,»r§="'-,_',:.,'-?:.?tK=r>-i<$,4,2z~>--.-,I-e:i‘'_3fli1_ei;2;y-7--r>.<--.'a?»3‘2.5..33/---\s-M,3i\:it,
i'<'-112%-2=1:§4'>1>-i:':1-:,»,;_-r;':-~->.>-..>>->2,»-r3,K'-;<.v'-éfifl.fi’)-g.’-'){;-?'’.=~.=,.;..»~,.--_,.;;):-,,,“.1‘-,->.'yY>.);-9’{/»1f¢-'a-J{Wi9.iiz/‘J°‘1;‘,-,-1,r-§>is?u>,--4.,.-?g‘:ilihatAT?'..t,-i-~2.,,;;-,,-2<J‘,,'2,xz-‘<>,>“X3-*x'»:<in'/:'/<,-<-j-.»s/._{/»--Iini;<‘,<;"i-i\i;y>,*2-,3I;;~..-,j‘F>51.%,’»/.'.,.?.=.?:£;A2;-i;§.\4,r\;,~'.,..‘.=;.i4;.1'{E:,;.P.i=i;=s-';c.'.-.i.i=i;-Jz;;.3g.=$'-Ye.5a:3F-.;§a=i-r1.}-2:-225.>:1-=-2-1E‘-I'i-a?;=n.<",-=.<a,''-:1.'.4=.-,'=1’5-?r.:--‘:215--.>"'I1-%.2_.e''.;ir5:'_-1<a>'3's1_;_'<:_.-=.j‘,-;_;>I,'=,.;_'=i.e'_=.';.1:<',-_';-"></f,i,i.Ir-:;='-..
-‘‘:4- 3,;-;i'-';--yIjg‘>,<,-*.<$§';];;;J,~-",i“._f:z'f:#.’?,?-i?f'§',>.==<-,-. .,=5_ .
;-=v=a“;.-’4§-¢re'Je-:r/,‘f-é=,/',§':i?';1f_,>-_=,=~.;j:<;;i../.,=,.si.;-’=~a>=é?.‘4--£>'.-,_5>>.;=J/-_/_-l;‘--E.4,i3.-.’;--y;r=}.4r?W»./-.i,=./$r‘r.\-§'.=TP~>\2§.;,-\:Z.’:1-?.’;"-'9-;;,/;r-:j3;'1»>i;'_r£:-=-,->-4r,:-;“-;:4.:3---,''<~->.?>;;1.>;<-,,\-..-‘4f.r=w‘-:yf45-£‘<<;<=\’>5-7>.=''4"-3‘>->_P§2§-9>*..9i.t,3',\=-?<''-"%..*=.-ift»2r=./,-'.-:,.-2-->»21'4:;3-€,-~.>,-=.K.~,-I'<.1-»,r_=;an-,',e4-i;=;-,5.:>.'<-,@,'?.=7=-=<1.42»',-=5¢%;-*./8,.-$.-;/.>X:..;,_»%~i3>*_-iT§=t_;-‘¥2/.25e~.-%-,%?.,1-<>.5-,;e,~§.r=.>.._1'>-g-=>%¢--';\,-f:.=:.;..»§'_Ji4-~@-=_;:;-i,'-€4!;;5.::,,__</;/,_;-j.y\;-.-\--*~..-;,»-.j§:.,--,1»_"»\.."-\;,§“.-;".{e\,.,-9.i¢,,/“’-.>.Q4<”-1."‘M-..i"".?,,-~-=l>"1;ii<_5:_.2.\?;,'I---.i'~,,?-_>s,6'-.-33L~.-;-1‘5.-.-i-;5>,-1§,1-?,=_>!i‘=.:.{,,-;¢-_i>,.?.<f,é§.;.r;<2;2r;.;,==,2-,'<-,.-=-_1..=,-:,i.rm.;»".'-,4y'7..-,2::-:-3r;41=--:,?»-.,-;:2..t=<§.,s,.i,@i;.a,.;;E,-,;i,.ra-_,e;s;'.,;§.:@,.;:_;,*;.?1<,,,¢.\-,-a2r§'-'r.;§&.a-i§-,€;§.4,=':21;.2I“_.;:';;;/.,:..;?.;-<..;>.,_-;.;:}...>i_>,;?;,,.g--.:.-;-,=-.i..i‘-;;-':--.>;'-?=:;>i;.n-f->;‘..-;--‘--,.,=-,f.'.,-.=i2-.’5;i.--->:t=,.-..:-I.;;-»=l3~1...-§»';;---.§;,;'-.5§.7_..,<-5<~3-.13;..;-,-¢1/--4..,;E2.;.;1-,-:'-'-_=3;=3,--I-_.’:-.--)-$I._.-2-,-.-‘f-.;'.--=.;’a._.=>,:-?Q-.;=.-,E--,‘-I}--}.3.'',-,-=-_&‘-=-;i':€-_.="-,=-=':>.f:;'z,;.i-,§I3r¢.:;
=-em;?”.'=’.;/11:.:-1.e..?:_---,;X¢/<.-3=-.-;:?*’--~;-,\'-.‘.=_\=,’i:'>'=.?;-'r,<.--_;;>.,'>~2.-v>-4'-_='i,-¢;.¢.-s.ir.'-.'_;-..=,‘_<=.\::'=z!-ff-.${i9,=ffi:.j1=§§Z£-1?;E;E,-,-EF555}fiaj=§.‘f:=?3££z'3-\>'''.~-5**'‘,\i-1;?,;-§‘Z;.i§<",}§.,)-'=--.;-¢,-;i,>~,5-.-§.-r.-E.,=»§;,'»,_<;»if;g--i.--'->-,r.-.>.3/-._<.:an~.2.-=.-'-i.=§§=;-:z_;=;=.j;;&_:;:2>;j-1=_:-,$3:>;2&(,"4;I;;'i;-?.'--:-f;r.;'.=..==.>I*---.-'st.i-»-=;,<.;=
‘="»“<-1-'/-=>‘:-=§“/'<3. =r---an=%;ifi-"z-:-=-eaa=-9-r-s;r-5:4.-_,,we=:2-.-mi-'.::2-:¢;z=rtfiitr:2eat=ansrr;*:=?=?*‘*?*@r§'€§ .3"ween.-. ‘-a~<‘5*‘>=~==~:¥r=§-<é=:>‘-?-;-r~::+9~"="..»$’.z~>
»‘>"z+=/.-5:1?’-X4-=»§I-,-=1 ¢~"_"-‘En/\:,>,.;:‘,-,:. _ j.’>-,»_-.5-';--.~_;;;;-;-‘,--'3¢2_{f2,\4:=‘-5'2},2-;;'{.'5£?.%@'=.~.F>"{5W".-,>-:i_=,<-5:,-;’,":.->;;'e?>->-:~',--.' -:.=_»'t'=;-\i;,-,»_._-;',»_.;-,=>,<-~:;'1-PQ;_...»->-.-7%,-=<_<-1-g1’".-.’>;=§:»:,.25-$-:-;;-~=
"e"'r<1-3//‘-J:=.5-'-‘/;£)>~<'~!z‘-;i''s1T:/-1J?/Q’-F7/f$fi>*,";-¢.é’;-r:?=?2J-"-%='9-i/K"‘’/-!='9-j£s=-=';=>r§2>=’-'-->’><<*1=~,--e;1s-u.f-2.;b'-$;->i<'~vf-/e.'ema;-§1-£-=E?‘’-r‘.-->=-.v>'>2-;=<>‘=’./’;:=i'-,'-:'5~2‘/:--F='>-;=A-1.>9'-;_-;<-Q::_r>=f':;;'$>¢>';=>>-<'=r-'=3-99¥1='--|''?32;s?1>%<>>i-<<.'='>'F€$.:==->}=:5'--v'-~':;’F>'fi‘-F4..-€/z<<?ni;»,>=~.=.-,-=.-,’,-'I-'?ii:-"--1;11>;('>==>1{'-v5??9'-"f43=$-~3>%*'F;-C1*""¥/"-"F>2¢ _2'>-fla.w-;.=/§;;.=-;-;...f-r"|';I-r£2'"{"_g.y.;i,-/'8=5=._';.?;f=/--:/;-'=:;.~.;;>»z;<.>v;:-t;a-
:t"r.-I->-3T»A=-»_/-'.‘/".ylT‘3',\-_‘>§f_j1"é\$=..&i-5/?x,.i-:‘>=é!W"'%,-‘'-”“I<-.-d_-\.-f<i.?~-g.;*-I:1-_-:;3,-i-‘>S§.¥¢'3t/'@@>;-r:<’-¥-»,-.t§:§|;-§<‘‘5=§;;;'l‘'<.?@>..-§:=..~€,f-7='?-<';3;,;--1§-fl.'-,;.-“§=7!>?:g?rs.{L'=?e=';>>‘'i,-[<'>."f:;if--§ia;'i9-Z§;.@=:f;§i>fl»:.:%5-J-;ii;s..~J§/-?'-;,;~r;:a,§§?-ji;/;i;'.=-1-.;'¢~:53=;%'~:2§,.$-;#.?>a,2=i,>;i-i--.?.J2:\?--F:;$;-,;-.~4-<-tw»-<-;E=,,a.--;Z-’§-,%;'F.z£,-'IiJ-_.t-e¢-.=1'-§-I;a;-''5-.:1,:;-‘;="\-¥,>-i»'s..$5-=%;1:t.-_*.‘,3-<§\,-;:r5;;=;’1::a.§;'’';?‘.=;:r-.»=L_.>=,{,(-=',;;=.=';>.;€-:r\1.=--.,.=2‘~5<.:;:>.-1.1w-;i-¥_2i>%i;-f_:?;,>.;-5¢-g??ym/:1-F,;-.-;:s'z-,?--E;?,.1;?‘~r:f;If;}-?-:11a;:>;:;§;iF'§=.-:¢r.:;-}?1,;=l.?-§.4£-f..;5-;:"-;-2'::‘.;£:a~.Z_;.';<'..;?~>"_;%-=?'-2>3--1=-_5-£:=-;';>_;.;§‘'=-f§§;-I-;.‘‘='.$3:>~;,;'><(,.<<=''Z;-.',._.‘-:-4/J-£‘-,3:2-';§.,'=;r1§;¢,i=_1€lr;_-t=ra'-5_5=;_‘2-=.'-2%tfj§>;.:-,_.-$>';.;3.-3.=;-,,>I=>€:=;-;,f2;aa;';:-s.5n-.'>.,2i-€-_;.-3-;},-g.=;2--:==-=PF,a_>E.i¢.'.-;,:;';};=.;r‘;_>1>£?';:-§_££€2,-§,;i_;:<-.'=/1£:;;;:§;=-:,3i5;$£§5‘§1;J,:M;5;-;-£:,:'P'i;i3-%:5‘rQ=,-,:f#-=7,;_?1£:=->.-=:;.;.<:'z;%-->{:=;=i.?;<>s><‘--{"§;a>1;<',£,_>-;>.a?'~=-jJ;'¢;Q,.=:;_y-;?§;F;:=,__}--&G;-'-e_=fiI,_;:s-m<E-=_41~>":'-afv;;.{§_;a5>¢2;;2?a.5;a-;:E§-'..-';r;?.<y;-<’';,,..2:;<,r--:=.1?;g'_@-s::.j:3-i.'e;_;;;;~-=4;:';r_<;~._;'-;:_:§g;-:.>;i3;$q-& ri?§9~i;:<g.~;-;,;=-;-.ig.i;>;..;-;,»m.=,'--;;
' »,.- _.,._.-,-,,-.--1;,-_.ig$!2(--<.1//'.,€\?-%,-x<,<<'>»€4,@>,,Z$s.-.,.-=.,_».\.,.§-h.-=.--9.,,.-:1..2.->._,,-..-,,.-4<~.\~_,.-_-_-,,~.-2;»---.-.-.~.M;.--.‘-.-.>--._,._-\/,,—.q..1-~~?¥:,>.:-=-»-..1.-5<2.;-,-.=-=&<1..%..ii,*--:,1.~,;-<m<~n:-»r.ii-'i.:*.-.'£..-w~t»:2.d“=:=;.<zi-v:-‘re.-..=-.;--I,--=.»-:1£r;F§F(’$=r».?¢'-.\$uu‘A .,.1- .,3,-...~,-\--2
Fig. 1 SOM immunoreactivity in the stomach of the dog. a: Fundic mucosa. SOM positive cells of a
characteristically elongated shape (arrows). ><200. b: An SOM positive cell in higher magnification,
showing a short cytoplasmic process with a terminal knob (arrow). X500. c: Pyloric region. SOM
positivecells are distributed in the middlethirdlayer of thepyloricglands (GL) on themuscularis mucosae
(MM). ><130. d: SOM positivecells oftriangular or ovoid shape in the pyloricmucosa. ><320
Fig. 2 SOMcellsintheduodenalmucosaofthedog. ThreeSOMpositivecells (arrows) arescatteredinthe
epithelial layer. Thecells aredistributed mainlyin thelower part ofthe duodenal glands. ><180
14 A. TANGE
E'_i_"-‘-_I;.Ifiii";-I-:3.-3.'§-‘;.=;;'-I_"
51-1-"'.=';-"ii?j-.j=}.;-.-<-;-.i -.
if-’;T*.;-=.;.5E-‘=j¥>'.="':¥:.-".-‘.==-'-'; I.'-.-"1.3--
__: _s-_.:H.3.;-; ‘
-.',-.=;';'-';§_-._;,=,'._=J§‘;?.'_1.%_;_'-:32:‘-.-,b.<e-i-2¢'-:-1.--'_1-:243i;3-</.g-'?u&ay;'’_3-t:*‘_1;>i3§;,--=->>.='<f'<;.--;;':¢.--a':-;-;33'=.5;:-'-:-_.s-'1:..-‘=.-_1--_5=--5'-,-;.-=__=;:-:r-.2-I»;2:-::-.. . * --_..:..'--..::-.'.'-2-I_5.21-_.I._ 11
2~;=r-I.=;=-:'->=.‘§Q<\,_icf‘.§'.';=--.'<>:»*l;-:,-;._-:-'==~'-34>-'€(;':-_:-:I;.’;'.-'.-.-;'-';'{=.;-;;'=.'£$:_I -I-;:_5;_=-j-; -- . - a
" -_-._._- i-i 3%X
z';==,=-_".1.<_-&,1.'.;-)-=-<_4. ---;-.;r.:": - '-.=_:_-=’,;:-?:,:>,',<j;<=..;5-=.-;--;'.-='=-.=:.'_-=;.,-_-_:-_;_= 're_-"'-;1‘.-.-:;-_>;;-:-:-:=- ::_-5-;-;--:;.-1 >8--_it--.;1=-;-.-_I-::-":: :-.--.-_2_':-_I_1'-';.-"I-2? .j-_I -;_3-.j 1 -§
I1- '- -.-. .' 1;-'
J fit _
y’ >:g>‘¢~ “”ax
" iiii-Ei'EI-I'-I-'3"I’:;-iii-3'1iii-'i'5'-'Iiii >I:‘=\-_i{iT:--i5:;--:i_I__i_,'2};-_I;I;-.=-:..¢>-.,1_:---:-;-'~s;<i?:-».;,*$".;'_"3.:--‘:11-S;'-';;->§2$?:ax1¥_'>:1=':==.''-?'.\'i.-?I'i=E5E.I'3'-'-I-ii-7i:?1:?ik-§1:§ii-iiiii-FF??§I'5‘.=E’z@ ' '- it.2,, -s\-,,
-.. _. ;-_'.-.-¢_»,g>;--.-:;-,.-----*=:\r,-_.=a;-j-;-- --;;~'-.-=--;I<>;:_-:;;1;,-;;=_"-' _' -- .'..'.-.-.-_1 .. i 3/ . ..
. :-:=-.1:--.2-"-1.;-.:-::-:-'-a'.-="-5:-:5- :.---1;;-.-1-.-1:.-1-:1-;f-=
-.- -.-. -.
- ---. -. .
_>;;i:,g;_;'i-,i-!,;.-;_-_.-;;_-;-\-.;-;§_;->2,"-;.-;.=;.;;3<%<,;,~'.;.-;_-5;-;_=:-_._-.,::.;_;_._,_-.;_-_ ;_.;_- _- ;';;. 2,-i,,i-J.__1.._._ --.. 3
- “$9?._ ; _- 1 Q
fig é
'--.'::_'-3--_'.‘-'-'-'I:-5..I--?.._.=;;,,.;:. ‘;£f-'- .'-,'.;\,'.-;;,',£.,:,’r--.I--'-,,'~,;,>.j_:.=’=_'-,_';-_-.._';.':.':=-_f-.:3.‘_-=_':I_-_':-:.- -- Q _'._I_‘.; --__."_-__._.__-'-_---".'--_3.-5a§.~:_n-';-.?::--.'
. ='-'E---'-.-.'='==:I-===-'-..'.:'..:-_;-;-:=_.-._;.;_.y;,_=_..j;;.;.?,;,-8§2._<_;;,-_-_-.--. .,_._.,_,;;.';;r;-:_'.:',?;.§:.,;-.;..;--j=.-;'-;5'-_.-.=-_:--_>:5--. ----.- -_-._.--' 1-.-'-_-..'-.'--=-4-.'-1- -I'.--__-- é$-
.-7>-:
5
Figs. 3-5 Caninejejunal wall incubated with the antisera to SOM
Fig. 3 Numerous SOM containing perikarya and varicose fibers in the myenteric ganglion
between the longitudinal (LM) and circular (CM) muscle layer. SOM positive cell bodies
with dark deposits on their cellular surface. X200
Fig. 4 Circularmuscle layer. Long beaded SOM nervefibers (arrows). X360
Fig. 5 Manyimmunostainedcell bodiesandnervefiberslocatedinthesubmucosalganglion.
X280. CM, circular muscle; SM, submucosa
in themuscularismucosae. In rare cases, SOM stained for SP were seen.
positive fine varicose fibers were seen in the Numerous SP positive nerve fibers were also
mucosa beneaththejejunalepithelium. presentin thesubmucous plexus (Fig. 7). Some
of the fibers originating from a submucosal
ganglion entered another ganglion. In the
Substcmce P Immtmoreactivity
mucosa, SP positive fibers were densely distrib-
Although no SP immunoreactivity was found in uted. They penetrated the muscularis mucosae
the endocrine cells, SP was detected in neuronal
elements in the entire gastrointestinal tract. A
few SP-containing neuronal somata could be
found within a single ganglion in the myenteric Figs. 6-8 Canine jejunal wall incubated with an-
and submucous plexus of the jejunum (Figs. 6 tisera to SP
Fig. 6 Several immunoreactive cell bodies (arrows)
and 7). Many intensely stained varicose or
and fibers in the myenteric plexus between the lon-
non-varicose nerve fibers were found in the gan-
gitudinal (LM) and circular (CM) muscle layer.
glia of the myenteric plexus (Fig. 6). Here the Longbeadedfibersrun parallelinthecircularmuscle
fibersformeda densenetworkwhichsurrounded layer. X200
the neuronal perikarya ofnerve cells. Some of Fig. 7 The subrnucosal plexus in the submucosa
them appeared to make an anastomosis between (SM) (arrows). Several SP positive neurons and
varicosefiberscanbeobserved. Aloosenetworkof
neighboring ganglia of the myenteric plexus.
SP positive nerve fibers occurs in the muscularis
A relatively moderate number of SP positive
mucosae(MM)andsurrounds thebaseofthemuco-
nerve fibers were detected in the longitudinal sal glands (GL) to form a periglandular plexus.
muscle. Many SP immunoreactive nerve fibers X130 .
derived from the myenteric plexus could be Fig. 8 a:NumerousSPpositivenervebundlesform
followed to the circular muscle where they a periglandular plexus and run toward the villi.
X200. b: A network of SP positive nerve fibers
usually ran parallel to the circular muscle layer
underneath the epithelium (subepithelial plexus).
(Fig. 6). In the deep muscular plexus of the
The fibers are continuous with the periglandular
circular muscle, especially thick fiber bundles plexus. X200
aWMW"DwQI\V{'”W”)HfI{m»/r""___________w_“’_fiH__H_____"_L_‘whuhH__H_h_'_____£__________H_______"__"____w_L_n__“)""_J_____"_______h___h“I_mr “"“____‘_yd“?______U_________uH______________ ____V$P64\I_’M_‘______H___________________3______'__ _ ~gg______\H__H__H___________H_________;_________§_______fl_________r_H_H,_________U“_____fl___‘__nH,___):___“___“h_#__H_____‘_§__________U%_U__H_______________________________"_"______“__U,H__z__U_H'_U___H__F____m__I___H____________L_m_i_m___Hv__Q___H_W__mM_i____HM“H__8____"___“”_H_m__“___'_________‘_H__________h_________”___“M_\___‘____'M___,_ h___‘___j_i‘iH__t________________1_’:_%x___H_mvH_H_J__W_n__J___!'”H_ i_____1____________J___w_____H_____________H____,____i___“__n______‘_________,_F_______'"_________________________‘_i_______________%_____________,_____N_W__"_1_______H__‘_____kHfl___M_f_+_____H____W_“"_H__'_‘gmF_FK_”H__’‘__m__£H_PmHn’Iflmh__v_____”m_“__"__H_____'__W__“H\____7P_fl‘_H___\__>____‘_H‘__mM_m__‘H__,w_|_b_H__I_H__~Mnr_H_£__“__W_____?w______"w_____nJ‘’__mPHfiWI__H‘__m_“_.___!___i‘5W__qWP__k__\____‘___av___________________________________________5H______Hmn_fl_£Him__H_“_HH___M__M_i__mu__”"_%_z__&_m'__d“_____H___________Hy___‘I__W_______A_T_H__‘__________}____9______x_.___rv________________(_________>______“__‘__5____m‘__F_‘___\______""____________________‘___6___?__3___:____h___)__________2 \’____I_________________‘___ __\aU___n____II“2hi\UI_gf_r_H___/_$____p__‘_9_I_________‘__“H‘___a________%Q__»_____________m____H___m_____________““__\_____H_J_______"“H______>____'“________'___h_"______"m___3,_,HuH_M_________HH““___“_H_%_r_____N_)_____;________~”H___{___‘/_“____“___‘_H_\____,I_$QIfaPI|_W_fl,;________2WU___Q%__3w‘?i}fM6’I(_}_W5j_Z\$__W6r"n\_m_mmv“_m___”/___‘_w‘Hé_“____v___H____h’hm__H___M_"_hH__H__r_H________HHm__H____H__HH__H_H__________m_H_MH_"_wH“H_M_____H_mH__‘fiAHmW_qM__H___V_H\__MMH_____m___m________m_Hm_______“__MM__H“H_""“$m_W_N'__"\_____m“W\“___H“_m_______mWHH____H__W_P__M_______mHm_H___“_"_wm_,__WH_WW_ww______"_%HH|HHH____WH__MpH__‘H___r“%M__fiWH___%h___;“__H_2'___pA_____________________~_Ji___________V‘____W__-_’I____'"_WH3_______H____H____a_l___l______%____I__HA2____;_r2____2'?____H_rA__’___,_)__‘_________.___________0_______L__m}”HvI"___H__Hm_g______H___"m____"_"____“__v“Hm___m__“_"’HH____"___m_"H““_/_T_w_R_H“_____w'm“____m___Hw___“"_hHflU__vH"_"__"_______“m“WwhQVm__wm'a__H"_:‘_‘__L_’w_I_f_W__A______"_,__tH___,___!_________)______/Nr_
_______________H___________H_"____‘_*______H___'_____H_
a‘_n__h_\‘yr'2/
_____flH__H____________________H___‘___q____h__“_"“_“"_________/H___H________H_H;b_QyM____\__H__H_____“H__H_____H__H_____WH_“HH_______m_h__m_________________H____)____"_____“_v____h___"____Z___““_Uvm_J___lm"“_"_A__H"”_“___H__l”H|MHW____"m_H_“m@/___"_M__H?H_h___m_“_H_______H___m“H_Hm____H____m:_____w___,_____________r________ __________‘_______________K____'__________H___fl___'______L_____H_J_H_________________h__“__H”PH_h_w_"_"H”__HHH"U“#_M_____"__v_mHW_"“___Uh__hH__
‘WW‘W_I__’_k 3___
___‘________"_‘_____________‘___I“____“_'n___"_____H______H_______I:____““__n_'__“H_____"__"__HW__fi__________“__J____“y_JJ_w_/__%n___m_“m_hH_h__W__n“_"%_“_mW_"h___'“_______H_____H_____H_'H_____“__
I§22 f_4qIr___|______3!_______,H_H__U____“_________m_H_“_m___“__C_________H_HmU__h_,“Hw__H°%______h_w__%_______WH_h__HW_H________M_M____Mm_H“______m_W____K_____Hw_H_____H_______“____h_____Hm____H___x____”__________________
_______3_""“___“%______"___FH)_|Wh___""A_m___“"_.____‘u_h"_;_P_@_m___m__h__H____,_y_U“H___{____
_________________3m_H___H__m_H____fl____"_“___'___T_H__m"______'"_____>____n_H“___H__um_____"__v“"_r___H__fl__I_,“___h__?"__________H_______H______"__H__”_:____wm;__"_F___n__H_________Ifl____Hh_____
WW/Y
_M__H_____y_3_
s’fJ/;s__‘__fl____UHU___m____U___H_______‘__________,____m“_____________HH__H_rm________H_____“_h_,______H“__H'fi________”“m,____“_>h___fi__“h____2"m_"_fl__”____<_________________I__H_____W___‘_n____,_2________u__H"___________>”__M__"_"m_%_M_“H_H_“__“__H___HmH__v__,___H_"_H_T_A““_fi________hH_m______“m__H__rh|W_______”__""__
______________/_
_hiJHmw_‘__‘__m“___H_\_"__q_'W__W___nh__v_>_“/v__vMM\_V__w__‘_W:"”wh"__w_’"?_H___:H__:"H___ _____________é____ a3X/AkW“‘/w_7_n
______y_'_
*_Q
vQ
F.1gS 6 ow
16 A. TANGE
from the submucous plexus to form the sub- themyentericplexusinamannersimilartothose
glandular plexus, and ramified around the bases found in other peptides (SOM, SP, VIP) (Fig.
of the glands to form a periglandular plexus 12), but only a few immunopositive fibers were
(Fig. 7). Thick bundles of SP positive nerve located in the longitudinal muscle layer. The
fibers ran from these plexus to the villi beneath pattern ofdistribution ofM-ENKpositivefibers
the glandular epithelium (Fig. 8a), where the in the circular muscle and its deep muscular
fibers terminated in a dense network ofthe sub- plexus, the submucous ganglia, and the muscu-
epithelial plexus (Fig. 8b). laris mucosaewas mostly similarto that ofother
peptides, vr'z., SP and VIP (Figs. 13-15). A
small numberofM-ENK.immunoreactivefibers,
Vasoactive Intestinal Polypeptide lmnmn0re-
however, did exist in the lamina propria of the
acfivity
ileumandcolon.
No VIP immunoreactivity was found in any
endocrine structure, but in neuronal structures
Neurotensin lmmunoreaclivlty
VIPexistedthroughouttheentiregastrointestinal
tract. Many varicose or non-varicose nerve NT immunoreactivity was found in both endo-
fibers were intensely reactive for VIP in the crine cells and neuronal elements in some parts
jejunal myenteric ganglia. These fibers formed ofthecaninegut.
a dense network surrounding the ganglion cells, Endocrine cells NT immunoreactive endo-
afewofwhichwerealsostainedforVIP(Fig. 9). crine cells were distributed throughout the small
Dense varicose fibers often formed mesh- intestine and colon. In thesmall intestine these
work adjacent to non-immunoreactive nerve pyramidal shapedcellswerescattered among the
cells (Fig. 9). The VIP fibers of the myenteric epithelial cells and were mainly located in the
plexus ran through the neighboring ganglia. lower half of the mucosa (Figs. 17d and 18).
Many VIP fibers deriving from this plexus were Their cytoplasmic extensions closely resembled
distributed in and along the longitudinal and those of the other peptides. In the colon, NT
circular muscles (Fig. 9). They formed a thick immunoreactivity occurred in the epithelium,
bundle in the deep muscular plexus of the where numerous NT positive round or ovoid
circularmuscle. Aplexus ofthe VIP fibers was cells were observed in the margin ofthe vertical
seen in the ganglia of the submucous plexus; section of the mucosa (Fig. 19).
someoftheirperikaryacontained reaction prod- Neuronalelements Although a few NTfibers
ucts for VIP (Fig. 10). Amoderate number of were found in the myenteric and submucous
VIP fibers penetrated the muscularis mucosae ganglia (Fig. 17, a and c), no immunoreactive
and joined in the subglandular plexus. They NT perikarya were detected. The nerve fibers
further ramified around the bases of the glands formed a loose network in the ganglia and their
to form a periglandular plexus. The VIP fibers varicose fibers often displayed meshwork which
originatingfromperiglandularplexusesextended surrounded ganglion cells. Near the myenteric
towardsthevillibeneaththeglandularepithelium plexus a few immunoreactive nerve fibers
(Fig. lla), wheretheywere scattered in a dense were detected in the longitudinal and circular
network ofsubepithelial plexus (Fig. llb). muscle (Fig. 17b),but no fibers were seen in the
mucosa.
None of the immunoreactivities described
Met-Eizlceplzalizz lmnnmoreacrivity
aboveweredetectableafter thecontrol tests, 1'.e.,
M-ENK immunoreactivity was found in both in the specimens treated with non-immunized
endocrine structures and neuronal elements. rabbit serum or with the antigen-absorbed
Endocrine cells M-ENK immunoreactive en- antiserum.
docrine cells were demonstrated in the pylorus
and duodenum. In the pylorus, they were DISCUSSION
scattered among the epithelial exocrine cells and
General Comments on the Gastrointestinal
had a pyramidal shape (Fig. 16). Usually a
Peptides
small portion of their cytoplasmic extensions
reached the lumen. M-ENK positive cells with Diagram l summarizesthepatternofdistribution
a similar morphological feature were also found of the peptides. On the basis of their location,
in the duodenal glands. these peptides may be put into three categories:
Neuronal elements M-ENK positive nerve 1) peptides found in both endocrine cells and
fibers and perikaryawere found in abundancein neurons; 2) peptides found onlyin neurons; and
ENDOCRINE CELLS AND PEPTIDERGIC NEURONS 17
..“::z-'f, f-.,'_.- 1‘ N . .. s;-f»_=;_=-‘-_'_'._..-.. 1.1.'>---__=.i.;i,;_:;:.:=_,,,"‘.1-_=",,a..;;1.-=_.I-:=a'.-§.=.=:-' _ _..i,‘,;1:.;i:{§,5',;':I;-.,i;I;'a'a_§.._9.../..;.,.1_'5./5<2“$_:"'i',.;'E,\_-”.'_e._'=.\J_jz,ji_.?./fQ.=E3<;}';';;-1;f.I-I-'=;§.5.'='§-I-f-11.!.1-_ _’''';'-5-..--1.;I-.' -I w:1".-.1.:_.'.'--
E;2i@f-";I.3_;'_-1.:-_7-3f'-=3':;;'=€'=-"'='5§Ii."-5=-‘.12-':'Il'-'.-‘F-.L‘=1‘-"/_1'-‘3-".'5-:-=:I="-'3-'.-I-5§‘53.'';<'T;"-.--.-.-’'j='-' .'_." -_ -"_3»‘I I \."‘5';'1'I'.‘I=i<’?1:-_'.Ir'}:_''?-§‘,.,'4->‘5.'1;i:€?}i=-"._‘5"._-5-f=f’)..-§.-._'5-.';.='.:/,?-"I(\'*1-"-'-E?§'.?.='I-‘If-/-.'-'-.I;3'.I . '.'-'..- >. _"’_>,2_,___
i.:.-'I.1;g5=*3;.:v5:i;;,'.=-..'1=-._‘-=.-'=.i’-I.=3.-£;1-5.'3.-1r.:;>";..-.I-'I_=.,-_'*.--_.."_-.‘>;L._?...';=f~;;''-:§'1'}.';'-:."l-'=f5."~'E5'§5§=}"\;'=<\/.-,..'"=;i'"‘H_YQT_‘__g§_“_E.,5'I1‘-i.=?'5‘'.:' I."I=.-_5:3’;-.' ,1"5‘$3.-'-1=<?'33'-'"-935-I~"5-’<,?§"' ,1‘i:1; \., -.e'=':3'*.E=i5'-Ei;.5‘=1"';1.; :.-‘1??<"/?§3'3'1 .. . '5
_.=---...-5..I;5-',>2;-?;._;=;»f:5;_;§_»"5-1.'-';-';:-_-5I-;;}I1-;-I;-_-',1=;;.-I_;'._e~,;=;-;'':._--_:=;=',. ,1».;-;-;..<,-_.-_-;._._--..)'1.:..._...'" __fi‘;r_e'.f;_'‘_;._>‘__-.;-.';,§-;.-_5-'._~-;;.;,;/-__\-_.,1/=1,-__...__-._,=-._.;:.;>._;_.,..:_\>_/I‘J1_,-:,f»-II-_\f-._1’,,~.,:-.:;._=.1-_9-._.5.-5;.=_=_;;.._=a.--_,>.§;:I.j.'._.;:;..i.f:“,:E;‘'.“:=::1',i:-.,>\-<5~2»6;£,}i&_:3-:;.it_‘)i;,i\1§‘';f\I;__:_",g5,a_l“'-/....-si-n.g\-\.-v_Al..e,| ‘*t;\-'r\;.\--._-,1;_.=;=.;;.;-"i-P<i\,§ .--.'..1.._..._.1~.;_;‘.=.I;i;i_?}.. ._.-'-.. ‘- . _-_.;;..¢.;.;. 5
,;-:1:i5:' .,_~_=c;:_=1-.';.5g;.;._..-;.": ‘-.'9-. ‘E-‘<35'%-'§?1f_=e€;;"_/:;;';;;..=-.,-;;4=1§§:;;--;:-I..-'355;‘5?2-.-1..'-.;';;-";.-,.;:;5<-_1I.-=.:-_;‘-I1;-;-‘1F_-:-;..-,--:;=\.;_;1-§;5§§§.=E,=E.=.-::';;---5;‘ > E“,1;_~!!. '-.
,. 3,2,; 2/,1: t<f- ’ 1 _ A .4
»;_,&_ ' g {<3s
I~ Aq’;;;§ 5 ’
if _ ’ :==">§.~1 ., ’ ..
\-'#5A1>/1ii! ’ .9“ ’2“' .r‘:.~.-..
1- i$,39! *1,,,1 ,, 1\ .-.'.-:;z1.-1;--..'
~..__._
_%,,,-.._,
*i5;li=;¥.-§=g..j?;'§;j-.'.>"IiE;1_5i :';:3:._I;;..__..:-':-:;_:__;-_.5:'r5_t--:I_I-_f_.;._:_.E\_"".__-_;._=_..E_:.-:-__.:.=:.:,5_.T:_£3_:::__.'_.__:_5Q;:.:__-_._:--____3=.-..._;,-_,.._''=_1;-'1’j.;_-_5:_-;.';3_-::_.E:';5;'-T-.1-’ ' “»_1'51I--}.I‘3-._.:Q.="‘lf:‘'='.'-.:551-.;§.',3.'j-"':§'-»2.3‘.§_.-~=r“\"...“~.~:=.Y.;'-..'.~:5.">x_{;'j':§“-;:;-:"=,4'4.1.1‘2"1j37..}...A_‘;--.;:-'-Ii;i",-Qi"-"-. ii_"' -.;,_¢v.1-';:"_.-_..i.§}:f§,{-;'§-:i=I-i‘.¥=§5f.'5I5{ii="3I31‘Y;5.=f§-5'1.-5-$=E§'£=I=.‘i’i§’§‘s§"§féI311-.’;;=1=IEi§ii§;§§&i§§;¥'3'-=.5i§i{.”':'lFI-":5:3%?.§I‘=~:5:‘;51'§'i.ii§éi=‘%5E.:I-I-i&l?£I=‘¥*? ~-.II-§'.=j"3.:-"CZE55-;-~_I'_?"'?<§'=".5=.?i-sE".1=-':.'-=E*%1--:-F;.j';I:-j-_:,_-I.;§,§;.I;-_?I_-.'§__f:iir-.':-=*-.-i_';.-P~_=j.;-"_".jIj:;:_;.;;-_;;.\j;..:_.~-;____\.§:5-_,';;;-§_;1\.;>;J§.__-_.:-_5;;;..;:'.._='5.:I__§;'=-"_-1.-'.2=I=.:fI'="2'-:;'I:':;*_.1 . '-4i'-~E‘'f~‘-'.=.---:2=-_I1-:;:L-'.L‘Y’?.I"!.'?.''T._.'.Z'.-;“-,_-_“_". '.§:~'.>.'.I:1
''3
.": '-"_:_'j?_Ij-.i_.? '* {rm.'}'-i.. _.._._._
._ _>r _1_:_‘ ‘ ’ i
i ' J
_ "-
--,' :1 --
' 13"‘i-iv ?.,,* e ' ’ " , ,‘
-. .=
,-.... .1, ---
.en); _- .. .;A.'1.;...;_;,‘ ‘-_-£3.-r_i,=.-.--\._-.;._|
y ' y N! 5&1,“ 'X 1 1 Jr “ J. ~r_ ,
4' "'l £3;1 {$1" ’ n
"-r'I_-_- .::.i?i-'f‘I".'_'; ' "'.;>._I-*»".:_.,;'1_=-__= :.:-1' .:1;.$2"5_ 2‘ :_j_E;___;_:_:_::1_‘__-___::__._:_:,_:1___:.:-_<_:._.:::::5?\::_,E:;;_____._____:; “.:I-T--I1:':3.:?i:'5 __ * .»,,. _'-__'-'_'_"__'_"__:__':-_.
.'.I5=.."'1..--=‘'‘.F,.=--l'=-1..:--'3.§-;:',-=--',;..‘'-.I1-I:;.-''=.4,I-=-=I.'111~‘;..A:,-=-§"=_,.,s1~,’-:=5»_-{-6-<:>-‘-.-:.=''-"5.'.,-:.,-.,=-_,.-'=:.“:-..-...''.3.-'5--;iI:-..'"---.=--':--':;'=:-.._-'--,.,..,r.=:.-.:_--.:-1_-_-;---'.;-._--=_.:3._;_1-'-.;:'"'_1:i--'n.'1.:="_’-;_;;--_'.-.-;_.;-I-_.':.I'--_'-.:=11,';~T-'-,i,r1fii»'----2if==»51,''i=.;:--,.-ri',,.--'_'-':---If',,.:~»~.':1..--!1‘I:Ib-=,=---;5y'-L';.--',.=;--r=';.--i.,.-;=,a-',==i;=&>-._':==4.:-;.,=7'-_==‘.-3>.'I=}¢'f§.:~,ni\..~~'..3=,-:1,'L--.,=-:,_-’-_=.,..5.-..'T,.-_';=;;:;.r.’-;-..'._-:;'-'.;_.5-._-.-j.__''<,,--=_'*.e->1.¢,';<;..-?=".-:;-;-:i.=;=:,-.;'=.-=.;'‘-.''a'._‘.-..-.:>5.~~:5.-'.=-__;/,$_1=?.;==.=r5-€-<-.'!;".-.?:;-..=;.;-£.-.!'-=,;’‘-..'.i;’f,=.=_¢x;!..':3.;-:5=;',.;_>~.F;-.1:-5-,»';,~.»;,-‘<--.-5.;.'=~'?.'i_'1.'-;,.=,‘-=="==:-.'.--=.-=;:/'';=--i-+5-.:‘-=,z:;:aI.'‘1;>1r:I-e*j,,=.1;-...,‘;--5§-;£_.§E-':.-I;,,=;-,_?,z:=,a;’>.;1-,=,z=--i-=;.'>..=;=..==1.,§;,;5_--1;'..='.:.41_:'...:,:-.:-,-;.==.,---.'-'-/,'.:i;.'=;=.¢.;=1,;-5...:>_>i=.-;_--;.'.:-.11-'>...--;-a~~:-;:.--;.,='.»-y*:£_.e'rfi-..:/,"=-'-'".;:,-z1:,:_i“}'5,:¢s_:i;,'_,:i./,:".r-,,1.".'..;.,_"j..:,---".--_-;*>.'"-'-..,-1.-.9>:;=2'>.~;~<;1-.-;,.;-',--;,;:';/'''-,>v.ii.'';\;',-.'-<--?-:.=;..-$..--;.=;'.'.,-I:~,>;;,;',.>..:¢--.,-‘-,J>l=...;f"r.»-ie---'..'>.$;Ir''.----.':-.'-.-.,:=c".;->.''11\.--»‘:..,.=..--.:':.-",:='--=F',‘..;.~5'-ff.E--1I_.=..=--;".?''2---l-1-';,§_1--.-?-;''11}='.--.-.=...,=_5I-.I;;"f:'..'-!_;:-.----_;:.:‘i:?-§L'-:.:'''r-I-g.=‘;;,'-;:'<:._>-;-,.:-,-:=;4=:r’--,,.2.-1-?,-_6-:'=_._',.-*._f--.;-:i;=z1.-'_-.-_:.'.-,--¢.::-_,._-=;=j?.:_;-,-1=:_'-I'-';;,..,_-=__.-....-.»...,----.~.....--~.,.1,-'.._.:.--:-.__~-:---.;'.I1,:1"_'-.-=-i,'n‘':-"-;,_;->1'.',::-._;z:<._.'''.,>=-.'1.'1i,_-.7_.r°1-§'.-,.J‘.'_.7>.=._~G(?.._.1»,i.'.;.f';i.i;,~’\..=.-‘=v.._,.=.--_s'=':-~<-'.1.:;-.;....~_--:.,'-..-v..;'.-.'§,"::-.;:._..:-;.;.=2~-.';.''-;--r:1-.-;*'s..1..j'f-.;-:-.-=-5-;_.i-.,:,..;.-,=-.-.1._i-.'.'=--=,%;4=_~-..',1>.:.r-.,'.,;-~.:,4j;,‘.:..!.--'i';,..f.-:“-;-I;--.;_>I-:-.'._.''.i-.'....'-_;.:-'''.:_j.';.;..:----.-'=.,''...-..-'11.-.-..-1.'P,.».;."4'-"_>>_.<,,-->.:’4-z.-.-'.,~--r_':f~----.,.,...'s'-.'.---.--',..,-.-..~-
. T»’1. =--':_-=-».:1-=__-='..:,:--'.:-=I'-:=,..e'1".-:'-,_.1<;'._:-7=_1='-.5=j;'=;.i-:===i-r:'7r..,j.;.'-.1i§-:=..:'.:;;~.;=.=="'.'.1:--_;;.-'V:,.-,-:,-,==',--.:I5-',<".;.-.-=:;=.-.I<-\'1+=;=''.-":=-;_"=2i,-?..'.1.'-~~».;;;f<1'=:=-*-;.<;.§;i-e'é>.--.=§r>.’--m1,_5.{Z-gi‘3r";..,;-:_.:-__--__';-.!.'-:-'---'''-..---'....---,-'--'.'.'.'----f..----_-"'.1-.,_1'I-:<,'--'-1-_»-'...----...\‘r*'11r..~.-'-'.-5'.-s.--2-,’”’'-a--\-"=\.';s.:<'.-e..,-~=:e';:»'_>’;-=-:.;;="#¢I,;:".:3==="-'<-1.’>-_»=.;1-<.'_'.:==-.-1.-._=1’1=--'-.-.-;'-.JI=‘=<,./1.;|=/-__:.=,,5'~.-1=-L_r-.7--r,-:,..&-_»:--.-;_.'--::-..1.,--.'__:-'-.-j;;I,-'-.,'=;..-=--_;-..-..;-..-_'-==;-=,.-'--.,':_<:j.=J--.j'a>:--=,'::,,;.i.,;»-.i'_,<-?<;12__-*'=-.,-;.,;5'.-.*1='’--.§3’3-'<".a/-~;I;5_.1-S;'--.---==-,.’=.~~-~-*'»:<..-.-',;=1‘.-,g-.>,.i4»(-f"~.‘1:'-‘i--e1s-,,-,1';i.-5.;'.'~,1.-::,a:i-==-i1;>.'..i.,1---..i-f-'",,9.-:.,~'<.~:\:-?~1.‘ri.1=,1,.{/-,->_f5*,.,,:»1==//.,-,1211.~¢<.‘.1=-;..':>;(.1’j;.=:;<=,2#;-r"-,I'i'—..'--5.;,-1.2,5;1I..3;_i--;.*;~-~--.<'-5_=f';:'24s=.;.;5_‘5_:‘:_-,=;~(_"-J.,’’."%:--€—;-_s,1’.:/<-;-4.€1-;1'‘:;,./-;.»=7~..-;;fi;?.*:.1=;.-<'.,:__§1-,.>7._-._.»':'I-»==”;-Pa..,’.-:=-.'5~=,x4=='=,~;=_‘,4,/3'.,'1-.;=>5-ir;,,-:".''-'?==¢,I._:;I.'.1;;=-,''3-,.a,=-..:=r.'.1.=-;''.-f-=-=-.’'=»';F-—':--,-.-i,_,-~-§_,--'=.-,-=.--'’-=1.,'5-.-:>-=_--.-:';=,_-.-.''.,.--_.’-.-.;‘---..-.-__--i.--'----'-,.'-_.-_--,:_.,"_--__I-_._-..''_-r....'-'*-.3M’?---_'=-.-¢=-_,-_.“..''---''--_-'-_’--_---Q_-_....'_.;.,'(-=”_.=-.'-3:---=-;-.;-.-':-:~_.-:''¥-.:-_f,"_/.;-,11\.i.,;'_?:-_'-..;:'',_-_--‘-"----'-.=...-,1_.-=--_-;;._~'_:.-<_~"‘_---;..-.=-.,'a-=:e:-'::=_-.'I'/-_.'.,__..=_--''.---\''-1;>-f.E=..-5.1-fi-j'J=.-5.._i=.'gi_.=--;=;.1j».-§$..,_-;.3---.:i£.-3'-_.---Y'1>..,-ir._;_:.:_='->--_:-.{,==5-..-=.»-_--=-_.;,:-:"-'--.',.:-;=:;_;_-=-i=_-=.=-~-7»==..m.->_==.,*1-'--=..@,.-~.5'.--5g-=-§1=*;=-_i1=r;--,-=,"j.,_2,i.=-?=_.--.;.;_'_-',=-=-i.-1/-I;.1i;='-,;.'=-1..'-'i='-i-1%-,=',..=1z.,1-1-_.%<'*;,_=--/.:;v.r-~:4f=.-,_i-—<_'-_i..1-=:.-.';-'=;1.,=:";-_.-=.<,"I»-*_—;=--.=’.,,"~--g‘j.->..’-.i>-»§=;1_,.;=__1~-;,-:;_§_,=;==.-=-_v<»'.:__,*;_i.:;H_'f=?-i~->.)_.~-_;¢—_..>..,f‘,-"i;-_-;rT.:'§.-:.?*':-.=""._,.=>i{,,1.»i‘,;-,-.gfi=.;=-.-:,.=._;';;4~Zi<'f;_/1!?.,_'=-._:5_.-»._1._..;-,-:g--;--._-;:,>..;,;.;<-.j_;§‘--j.-_.i.-_j-,'-:;.;'-...;----:';:;..%__;.r;,-=!'=_4:I-._.,_1-f:._./t'';1-._{.---;-,"'1,,..--:‘_-'_;,¥;,-'5;--‘.1iJ;.=-:-'-51-'._.=,-,_--_I1.'_,-._q.~¢,1~_.::'''_-§>-_-:-'I;4-.---:‘'..:.;-_'u;;--_:;----g--.-5>~,':-'''f.,.<‘'I;".z1.§-,§;f',<3@-‘-.i='1-'._—':;j={'.4§-,-,.:-‘.,3--;-;.;:.ij1»;jZ,.';;i,.:£,-.i1__-__;§§'_5'-A,z=-;J,_.-:,'_-,d;':.*._';-_'j_-.',,:-:.:._,_'...E-_''_:.;,-;i~.‘-.'.-'.
,f'.-:.-_;:;'.'."3j-'.'I_If~i_,';';5.'-i'§.-".-i.§'.'i:':k=?<-?L=‘;i.\r.;-;-5'I3;'_ -..' -. I..'=.::'r--'-$53":-._I;:-{~.:.'-§=j.-\£'_-''2r-.;_1-.;:?_-.i-...-§~_I1I;-».';.'.=i_¢,;=-=:-,.'i=-f§,=;:g‘>,4:r-=:;;;=3/4;£<»;5/-.,—-';--~..‘i _=.'.".--\'--='-;‘:T;£ia;'’-/'Q5-'i",'-=.''-9",-,~'._'.--1'.:.'-._-2-'-...'--._ .: -_ '==='.-.>->'__.'--._2:.§_. ._--__-:-.. ..'-....'-'/.'-..-.-=:v,.-.<>'-.,-..-..-.-'>:.--'=7..-..-:)-‘-'-=-
_;='. ...-:;_.:;:;_._’/,-¢»..a_-.=‘.:,{4;'..-'_'_,_4-ji;:;;--._._..Eg.i&_;. :-.-;_.'; -.-.,1;:;i1=:.--..'--..3_,---:;;;._j:-;;;;.;''-.-5_-..-‘.;;:-'.1._--I_;'-''=‘§>§$; %'i?£':i#?;5f3‘3.-.<'i._-1:,';§;;;‘;==.‘=§_l.:~=$»l%,,€»i%1.‘?.€.>?,,;i‘i¢i5;='_E-.’==-‘<=_=.-.i-1,5ii'>i.,f.¢<;,_:?--;:‘;-->‘'-i'm,:-1=<;‘'=-.*.;==.i-:;-;-1b.:'i.*.-_‘-.-=_§=.-..;:_"5P,-.-,:_-?;,--_...--.:5_‘-,-1>-1;, 1--. ._-.rI-:'_-_-.> - >'.-" /.15ii»-..--;.'--. -i;;-_- -.-.;;--...;_.1.;;;1:1.15.;;.j;1';-._,=,.,‘-’;.-.;..:,-;;r;-2=’_Q;,"~,>;-.--.'.'._.-
;f,_ : _:;._;.;;j_;:,;.-_._;.=-; ,}_,,§;-1;;-2;.--=-<i,"£;;;;:=,->/;_'=;;'_'_-_.:.'1-}5;;§;?_};§:=__§_-I5-,;';;§'.1 .-,';I_=;...-;:;;-';".;..§'§-§..f,' ._'-'.-{T-':;..<,V’_ .._ -‘:§?'-_.=._1-.1;.__'--.''_''.I-.:_:___-___ /<~.__»;_.-_:_;';;;I.'-.I_5-;_
. I .-
..1."-‘<'.-*~".=-:=".;=.'--1_'-.;...=;,;;=-,::,,----I'"_4-,,;_!;;=.<_,<4;-;fi-ié.a,.,.1.,g,;$.<";==_=-.<:_=;..e=:.-:.-j.=.=¢2-;:.;-="s;¢-r=-_4-.?_-,,_-..;1;.;: _-= _---.=I»-.-,;;:-=>2.’-';9.. -..--:..,.1,__:-.-_,;-1- _ .-.4.' " -. ..;-,1-.-._,-;~'1-".-." " .=;_ ,;_' ' '"-.'
E$1‘1".1I:-I---.;...;.--.-:'..3-=}1..=.I-.’-/§'W.(1==%~.-.i==;=>;-<;£-=.=-?.'.z'=-¢::~$"r?;-‘'§/:=.§_-.-;---.-..-.-..---.-;==-.-.-.-:.--r.<§=,;,’,-.5-.-';i..=’Ck;:£5e‘‘=>-<,I,=;:--.,>¢-=...~/1/9;<<{}.!-2-=1=.-‘;=t>;x:/>l£;>~,>1{/1i=;-;>)-=:{.-;’:-,Kz-1>-‘..>.=4,;=\1:--=,-,-.=-1,-..:.=-.-.D.fi;g--,.,’=.-_;d:;9--:;y1;,-.::r%er..=-=.'i-.*~..;--=.--.f-:r;1=.=-.¥/$Z1-.-é’.I-n:/"1:-;>I'1*“;:‘L_1g;.rA‘§11:'1J5A-- -.?--'-A4.-_--JIfi’2-’-I-I,'‘-'-":,=<;-='»*M‘11}‘,.§"..=\;€-.-.i-..fi.:--;;‘.\si\”,Q:-"‘g.=--\'M-=.~‘;5>!\>1>1'M1’1/,1,}./I:,J/,.1P-'::/-.3J.-=.=;
' " ' ‘
}-3'5 in; 23/ 2,2;/»< 1’:5* ?>;(s.» £3" ,. in YI“ r > =.<£=.7:1 "§\'-;.'_=.
--'1:-J» 4-'1.‘¢.i=ii=.135‘I<3f?i’?5I-'-13.";'. ..': -1if--..._.-...»,..../M: ..1 .=_ ...i¥.-- \.,.. ...1=--.-=:i ’ ... .. '', ,'' '
:_-;)2-:5-:5i;ii---n-..5-'=_.'I..§;;:=._'=i_:i'..5,-§;‘-i..:-:-;;.,si!.n.;2'--_:'',.--';.--eI:1'--iI.5a1:'=,!._E-':.-£3;P’;-1?''~.._-.;=".l='"i"“,:'1=-,.''-4<:..'-<*-=---I.--I'';.---_1'..=..5;~.'.E):=-"'..=-.'-:_-'-.-="x_v--":..=-'.-.‘-=¢"j:-*z;’=-=1‘=-'‘1.4;:13.’-".'~;,.ii’-",-"2--.-I.1>1.'":>r->_.>=.e;__--1.,-::3?.)1lg;’,2'->!;;.-.§_~..@E=::'==_.f;iz>1:g:_<{_'r;‘-';§:,.1‘-,&;-;'"r,=:,-§:,;.g:;;_;f;;:2-.4:.3;;.4:.--r3l:r§5j}=."n?-i1>;'~:;a§-&a;"?-;.<;=g»s;;;-2\;_@}.§?.;>,.=.2:-a,,"1¢-.’-;;...I§--?¢-%.1"§ .='.:_--..:.-=..=-I;?a.-;l,§-r.r*e.<.I:‘""§-"i-l}:'>=>I.'=';/r';}.:§_:-,Y:..'.§*??='“_=.-.:-==5>',=./-11..-;-:;3/_---E>;£:>-.;fl.-.-;gZ§..-;;g1;"-<==L-;5.---_=;=....-=.._..3~-T=--’5-._i£';<'v.§-3g.5ieg5:5:;:=§.:§.;=.,.=:.Iz..'‘§4;;;?<.;=.-;.':.=1§.-l_§-._'-r_;-{.;=¥;¢.=-{.E:.-:rg;-,I;<-',-,-j=;-_'-»;=§,{-,.--,':=;.;',-._-='=:-I-_'-I--..;..:-;;-.:..';;.:'-.;..I.--.-:;:-.,-,-;-.>=-»’,_‘-*-.»-,,1.-..’,=4.14
‘.,.,>,. . .. ,---'i;5'=@=1'.:i=:j;-.-I."'_:.-i'.;=-fr';'.'<...I;’r-’.'..-f=a‘-z-’:-’i-1%-:.-4'/.fi::=._-9-‘<~4-.-=r.<'-;-":-~a‘:-.'r9>i:’-~.;=;*1-=§.’»/{.-->»-.,@-:».; 3.?-.rearg-a=’z;-,»é' " -.;-.--,~.;;-2/'_=.-_:;-.-.- :.;;__.- .-;.. 1, . , .... ,,....... ... .... -. -
1.-,1/.-1', ..1.-».-,--.>_;>.-1..-.;--_1=.-<A.-._r1,-:,:§-3=-;B,-;'.-_--._...-_-:='.;_-.;.;~-.--.-.--..-.----i-n.‘..--=.-;;-..---.--..=-:.=;=>1~i.n=:-..>=.<:,--.=;¢:-->w-.t7:--/;?>;§=.<<=;~e1r1:=r-.w\'-a_==z.-=:--:;-.a;:n-;.;-:..:--.<i-=i=-a=;.=..=-.-.:i.=>§_;'=_;-._-r:.I--£..:'..r?=.§;;_;.l.'1=,-=.;\-_>-.’§.1->‘'.h1.-i- ¢ » .'.‘!-;.-:-r..-.-:-J-.-_1=;=n=1..-'-=:.,.if,- -=;.1:=.-;-'-.r-.-.--.-;>:.-/5'91’..- ;-.===-==:.=--.-'-=-..-;»~»-..->.-)>,§<@».-;..r=--i1:/‘,---..=-¢"..=.-=.--.--.=---.----.----..;--;--.-- . ;-<.~:._-:
F:-'M1,:9-:‘>;',_;,=>.;=>..~_/=1'_;.-__-'.;-:__.--';_‘.-'-_I-_-':_-I-_...*__:_::t-;-=;;=:;1.:-=.-;.._-:--;.;.§a:,-’..'-.;1;>-I5';'.a;';=-_..-2.-1};_--r;;.;-,-._1,';-_;;,;_=_.;.-_:_-_;::;..=-:-;;:-.-.;§‘éF":~E»$..}£_1¢’.'=::-',<>_>,-‘Q.-:.-.-.-.'/_,;:;i?-. ,-.,'-',~;.E-;5;.,_,’=.,-;,~r,-.I/5.-1€'2;-_?Y..<';:._f-’;<-1;*--.'-<';.;-;_;;.=,:;;=:'=;1§"'3i=='=‘'-1F"-''31-5''? -¢/,4~_-‘I.:§=a;_}.==,-_;:5;'.;2-;,-=.;..=}.1'.>-',.,_'-.48'17.--'-"‘:1fz-;¢f-'=§;-j,:_;--. -.=,/',==$5.-;_-,-=__:_-=.j-=.'-;'..1_.-‘.,=¢_>s_;-3'-§'=§'-'._==.j;a'._;§
-._.;f/ »--., 3
gr?‘-".':.-_"_-j.1'1"-,1-.§:1;;g§§I;;-';-.=I-;-';..I-I-'.'§.':':-:¥_I-.iZ§1'"'§§.=3Sa'.;‘=I'I;'l ,',=.I5;-j-.§-.j=I=:1%i~_>%€_.¢?“ 3_"_Ij;-III’-""3-j-iii=§"§1-TI‘I_"§§£§;i=_=.'z'¥:'._,.'1:-"'"='_ ‘4,.
,-.._._1-'-1r>~,.~'.?;=.-;:>-'~1"-.-'1_._~'.,=1--1=,'<~.--/.~;.,;-Fz‘.'.-',;.'-=,--..-:---_-:“.fi_-n;..=:--.'-;5---.-:.<'..;=5-:6.2->'::.:=:/<.:11;;»%_..1é-..1'..§\_/fi/1§:‘9J,1>>3,-.>15-;,.;;'‘1‘;y;;.%.=..>'€->_~::-',y-;,4;===,‘=-;'.=s',:*§,5-~'..‘<-_~_-"5,’,?;-;";'4<.J‘-9'-,'-s- 1=-~y:>'i.I'"ii:i.;‘;?!~‘;1,*‘J/f1{’§i!-.-’,:-1.1-'_,.¢,=-.*._’-...'-.._--f.=_;;-)>,.:-:1_-_-.1,4~>..;'g-='1'=-;i.5_.I.-.i_.=.-'I=;1.;.'E-.,%:.-l;E=-==.-.==:--:F-.-:=-:1i3'é§_i-5.:-=::-==.-':':':'=E=-F!E_5==:_'|-L'§’,;;,:-.=1;_E-.i1i=§L.5=--.=..=:;_'--'-'-..--';.=:-;-~:-,:=_->‘-“.-.'i:=5=;I3;I'_23r,‘=.‘.--:1,s-_''.-$.’..f.'>_?==;I-.-'.-n'ee2'\''_n-2:;;.=.{1.;;.xI.;;,.-\;_,_1=;<;j,;--;=.--."-3_='-*.-,E-_5=-='..-_:':-''-..._-.'.---.II;.>=.I'-.;';--j:.=-1'..-=f5'-1E-;=:-_.;"=-"Q-=.1'£-'5=-"1,,:-1,->;;'-1.;-/;I-.~~;;=.*.9.<./.‘,5>,-,1-1'55;-.-'r;-;‘1:.-\_I';.1'-%_..5’/.1-?';-1g2’>;>.-'2i'r;_'§.I-"-;;_é-=.j;>;:;-,_';--I<_-.=2'-=I9_=><;;,I-,---5=‘=:1--;'--'1r1.‘-)',;">'-2',=-.--r_;';-‘:..=:.:-'.4::;E’;3;:.;§__.=__-;.-.:==>-1.-;=i_;_,,=’.
Figs. 9-11 Caninejejunal wall incubated with antiserum to VIP . _ _
Fig.9 VIP-containing nerve fibers in the myenteric plexus between the longitudinal (LM) and circular
(CM)musclelayer. Longbeadedfibersruninthecircularmusclelayer, somecontinuouswiththemyenteric
plexus. Varicosenervefibers can beseen in thelongitudinal musclelayer (LM). ><200
Fig. 10 VIP containing neurons and varicosefibers ofthesubmucosal ganglion. X400 _ .
Fig. 11 a: Long beaded VIP nerve fibers form a periglandular plexus and run toward the villi. >-<360.
b: A network of VIP fibers underneath the epithelium (subepithelial plexus). A fiber connection can be
seen between thesubepithelial and periglandular plexus. ><360
18 A. TANGE
I __ _
'..
-ea.
31$:;_§~ I
we
01i‘1an* rni‘ -- anA
_ :;;:_-.;=_-';.' ' ii21 ea _,‘-K»I§ .-1__:;I_;-_.j;§-_ -,
‘Iv
Q‘. I
-..-:-.I"I I.'I.3.-I_:I:-I.:I:Ias
.__\";---"I3.- \-
a-.I3.-IjI''.“
ii ,_
'-
- ''_
i
_..:-.- ''--;'-' " § -211:;I' II I'I' "Iii 1}‘=:' ' III .
" .- ' '
8'
e>-6I:--.‘F=
IiI=3?.'I;_?w;IIj_I,§.;;-§,‘j§._f-‘at
I‘ "ii.-.<;z-:=.-1-.2:-1-_&E-r-.1:;-'_-'-- ='I;';I.'._'--. I I-1-I---.-.-.-7 IT-K I- ' _I_-_ -' I ..-. I -_H- ---> I'..-.12-_' -=..:.;.;;:';=4=r,! ;'-.::.--...--.':,... . tI”-.\=v,;-..-,e§¢.,-_;.--_ .
_. _-- ,5,,;,:fi1,:-:~.-..-;Z_ia.-;-;I_I-_.=..."_z:-I'.';-_I.-_-I;-_I1..‘1'-_3-.I_'- ' ._-".-II};-".;-_I5"_--__-"_.-._._1_ -._ ' . £E-I _1..\:E:__. 3.‘-I -
‘:,._._E6-;€5_-::.___:::_.;-:__j::,-Q_=;r_:;.--::.:.;8-:-,.?:?1- I":-sI-*_2 Iy3.."':--*'I3Ii1=-'::1:-2‘i:?-III: ..._ .-I..--:'--:.1'.'-,:.-'=->:%2’.’:r:-E.i-}-1I-'-f'.3'.-.'==.'»"$'§1.--.III--1?'-I-f',"''' '-III-11 5 . . -I;-;»F'="-;;?";‘'&-="r-:=.<'4‘%=->=';;-::>-€-a'-.‘.-..=-z?§¢=?-;.:..I’-1';i-fi>?';.-’I/Ti
._.: "I-.--_.---_'-._.----;,--.- ~;.->3,-1---5-._-1.-.'...-;;,-._:,;.-.,---.'----I '.)_-.I.r---.-'.-_-'..;---_-'-;-'.-.-;=---_.';§',;:_..'rA_\.f-é;--__--.-"-_-..=.._.'.,;_.._---,,.--.I-_.-_'_--_-__-__._ . I_-_.:--.;-.--F.'_-'-.-:-;'1.:_-§;,I-.1.;~:.,.,;.<.¢=!>;.=---;.-I-::.5;--{_-':';=_:i'x:¥-‘r.-;1r_,=-;::=z-IJ;r;.;.=;-'j1.==:-"§=.;'=f=>==-3:.~;_.,:‘'=:j-,»=...'>_'-;-r>.-=.'’-~<-f=2I.--..-:-_I-.I_.-.=.3-1-;;ér:---_.---.---;-'_-I;-..---_-._".--.-=./é->=.1=.=--..-1-k.-_'w-..==._...-..;--'--I15-;.-_;-'-_.-..;._--'._'._.--.:-_..,'_,>1~,..<?‘5.-:-;r--?3'-.';-:I:<§..‘,;'g.’_§;::;:_:;-f)>:"-;-,i:-';'-;=,'-;<:''.._=.j,-';_>,,-_-'.§-_-.;_-;-=_-=.;'5.;-1;;:'1,-.;;-j;-;.'';;.';:-I.,!=;==:1:3--.:E__a:¢aa.;!;-:1=::-=';=-=
I".''.'-'.’.--1'.I3_ ;I" -3.I:-.315';'_‘I-.5"}-‘'-..3'3,.3.A'_.,.5‘'..'-iE.=I.3=I:_1=i:''3Ifii"2n'.'5;.I5-I-:.'"-.-'-I'".'I'II'fI???-'=';§".'-'-.":'.-''I -....-'I--§.;-II-?F=.-'-'-.'I.'-'II---I -. -II‘'-'' 'I'-'.'- - I3.'I3-'-’Iit '.- 3- '-'.'.' ¢"I.,Ij'If:'I'.'3l-5fi_I__-.}1'.,-.,-;=-.=I'."I=-'3;:"‘"E-=I-‘:-
'. 31>;-I-_---~:;.';;.;. .- .j_-- I. 1--€-..;>.-.;- -----.,=-.---:1--./--;)1 .
-I I - .-
- 1 - - -."' -- -.-"- - -. -- w7:-_'_ -
Figs. 12—l5 Caninejejunal wall incubated with antisera to M-ENK
Fig. 12 Aganglion ofthemyentericplexus between thelongitudinal (LM) and circular (CM) musclelayer.
M-ENKimmunoreactivenervefibers can be observed in themyenteric plexus. ><200
Fig. 13 Long beaded M-ENK fibers in the circular muscle layer running parallel to the deep muscular
plexus (arrowheads) wherea thick M-ENKfiber bundlecan be seen. ><200
Fig. 14 Submucosal ganglion. Several M-ENK immunoreactive somata (arrows) can be seen. Due to
the thickness ofthesection (20/rm), only a fewcell bodies are in focus. ><200
Fig. 15 Varicose M-ENKnervefibers (arrows) in themuscularis mucosae(MM) beneath thejejunal glands
(GL). ><200
Fig. 16 Pyloricmucosa. Two M-ENK positive endocrine cell bodies are in theepithelium. LU, lumen.
><250