Instruments Used in the Measurement of Rigor Mortis

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Instruments Used in the Measurement of Rigor Mortis
9.
I N S T R U M E N T S U S E D I N THE M E A S U R E M E N T
JOHN
PURDUE
C.
FORREST
UN I V E R S l
OF R I G O R M O R T I S
TY
Introduction
The phenomenon of n u s c l e s t i f f e f i i n g a f t e r d e a t h has i n t e r e s t e d
p h y s i o l o g i s t s as well as t h o s e i n t e r e s t e d i n f o r e n s i c medicine f o r over 100
y e a r s . Within t h e p a s t 4 decades a v a s t amount of r e s e a r c h h a s been conducted which shows t h a t t h e t i m e course of r i g o r m o r t i s i s c l o s e l y c o r r e l a t e d w i t h many of t h e post-mortem chemical and p h y s i c a l changes t h a t occur
d u r i n g t h e conversion of muscle t o meat. The s t a t e of t h e muscle at t h e
t i m e of r i g o r development v e r y o f t e n has a profound e f f e c t on many of t h e
c h a r a c t e r i s t i c s of muscle t h a t are of economic importance t o i t s u t i l i z a t i o n
as meat. The s u b j e c t of r i g o r m o r t i s and i t s importance i n meat s c i e n c e h a s
been adequately revie-ded by Bzte-Smith (1948) ; Bendall (1960), and B r i s k e y
(1964)
.
From a more b a s i c viewpoint, Davies (1967) p o i n t e d out t h a t r i g o r
m o r t i s i s one of t h e many f a c t s about muscle t h a t must be i n c o r p o r a t e d i n t o
any t h e o r y on muscle c o n t r a c t i o n . Therefore, t h e measurement of t h e t i m e
c o u r s e of r i g o r m o r t i s and t h e e s t a b l i s h m e n t of a c c u r a t e s c i e n t i f i c p a r a meters f o r i t s c h a r a c t e r i z a t i o n a r e b a s i c t o almost all f a c e t s of muscle
research.
A c r i t i c i s m t h a t can o f t e n be l e v e l e d a t r e s e a r c h p a p e r s d e a l i n g
w i t h pre-rigor and p o s t - r i g o r muscle i s t h a t t h e y c o n t a i n no documentation
on t h e a c t u a l state of t h e muscle, nor do t h e y show t h e t y p e of r i g o r involved
( f a s t , slow), because r i g o r w a s not measured.
Defining r i g o r m o r t i s
Before a phenomenon such as r i g o r m o r t i s can be measured it must be
a c c u r a t e l y d e f i n e d . D e s p i t e t h e tremendous amount of knowledge t h a t h a s been
accumulated on r i g o r m o r t i s it i s d i f f i c u l t t o g i v e an all i n c l u s i v e d e f i m i t ion.
Rigor m o r t i s i s g e n e r a l l y recognized as t h e s t i f f e n i n g o r l o s s i n
e x t e n s i b i l i t y i n muscle a f t e r d e a t h . More s p e c i f i c a l l y t h e s t i f f e n i n g i s
due t o t h e f o r m a t i o n of "permanent" bonds between t h e a c t i n and myosin
filaments.
It i s known however, t h a t under c e r t a i n c o n d i t i o n s muscle can
complete all of t h e post-mortem chemical changes a s s o c i a t e d w i t h r i g o r m o r t i s
without becoming r i g i d and i n e x k e n s i b l e (Davies, 1 9 6 7 ) . Recent work by
Kushmerick and Davies (1968) s u g g e s t s t h a t t h e development of r i g o r r e q u i r e s
t h a t b o t h ATP and ADP c o n c e n t r a t i o n s be reduced below a minimum l e v e l . It
seems l i k e l y t h a t an a b s o l u t e d e f i n i t i o n f o r t h e end p o i n t o r completion of
r i g o r m o r t i s w i l l be i n chemical terms.
P h y s i c a l changes a s s o c i a t e d w i t h t h e chemical changes i n muscle are
extremely important, however, because of t h e i r r e l a t i o n t o some of t h e
10.
economically important c h a r a c t e r i s t i c s of meat. I n a d d i t i o n , t h e p h y s i c a l
changes i n muscle have t h e advantage t h a t t h e y can be reccrded and i n t e r p r e t e d as t h e y OCCLU" i n t h e sample, whereas most chemical determinations
r e q u i r e a t l e a s t a s h o r t d e l a y before t h e r e s u l t s a r e known.
P h y s i c z l changes t h a t o c c u i n post-mortem muscle and a r e
a s s o c i a t e d with t h e t i m e course of r i g o r m o r t i s include e l a s t i c i t y ,
e x t e n s i b i l i t y , l e n g t h , t e n s i o n , c o n t r a c t i l i t y , e x c i t a b i l i t y , hardness,
sarcomere l e n g t h and molecular c o n f i g u r a t i o n .
Elasticity, Extensibility
The e l a s t i c i t y , e x t e n s i b i l i t y phenomenon i s one of most u s e f u l
c r i t e r i a f o r measuring t h e time course of r i g o r m o r t i s . T h i s nethod t a k e s
advantage of t h e e l a s t i c component of muscle. When p r e - r i g o r r e s t i n g
muscle i s s t r e t c h e d t h e e l a s t i c component w i l l r e t u r n t h e muscle t o near
i t s r e s t l e n g t h when t h e s t r e t c h i n g f o r c e i s removed.
The u s u a l procedure involves t h e loading and unloading of a
s p e c i f i e d weight on an excised s t r i p of p a r a l l e l muscle f i b e r s a t r e g u l a r
i n t e r v a l s . The changes i n l e n g t h of t h e muscle between t h e loaded and
unloaded c o n d i t i o n are measured. Data can be r e p o r t e d as a percent change
i n e x t e n s i b i l i t y , modulus of e l a s t i c i t y , or t h e e n t i r e recording can be
i n t e r p r e t e d i n terms of l e n g t h of time t o onset, delay, and completion of
r i g o r mortis.
The f i r s t apparatus of t h i s type (Bate-Smith 1939) recorded l e n g t h
changes on a smoked drum with a kymograph needle. The weights were loaded
and unloaded manually a t 8 min. i n t e r v a l s . L a t e r t h e apparatus w a s modified
(Bate-Smith and Bendall, 1949) so t h a t l o a d i n g and unloading were accomp l i s h e d a u t omat i c a l l y .
Briskey, Sayre and Cassens (1962) r e p o r t e d t h e development of an
apparatus which r e p r e s e n t e d a refinement of t h i s method. An e l e c t r o n i c t i m e r
and solenoid w a s used for loading and unloading t h e muscle a t 2 min. i n t e r v a l s . A n e l e c t r o n i c sensing device was used t o measure and r e c o r d l e n g t h
changes i n t h e muscle. T h i s apparatus also allowed f o r t h e c o n t r o l of t h e
temperature and gaseo-us atmosphere surrounding t h e muscle.
More r e c e n t l y t h e Wisconsin s t a t i o n has developed a m u l t i u n i t
i s o t o n i c - i s o m e t r i c rigorometer (Schmidt, Cassens and Briskey, 1968)
This
u n i t can be used t o measure e x t e n s i b i l i t y o r t e n s i o n changes i n up t o s i x
n u s c l e s simultaneously, all h e l d under i d e n t i c a l (aqueous or gaseous)
environmental. c o n d i t i o n s .
.
For my own s t u d i e s I have designed a r a t h e r f l e x i b l e apparatus t h a t
can be modified t o accommodate a wide range of i n v i t r o muscle s t u d i e s .
F i g u r e 1 i s a schematic drawing of t h e Myotron as s e t up f o r determing t h e
t i m e course of r i g o r m o r t i s .
An important f e a t u r e of t h i s apparatus i s t h e p r e c i s i o n w i t h which
t h e environment surrounding each i n d i v i d u a l muscle can be c o n t r o l l e d . The
p l e x i g l a s s chamber i s jacketed and f l u i d from a c o n s t a n t temperature circ-Al a t o r (Lauda, Model K2/R) w i l l maintain d e s i r e d temperatures a t -k .02O C
w i t h i n t h e temperature range of -10 t o +150° C . The chamber c o n s t r u c t i o n
11.
allows t h e muscle t o be suspended i n an aqueous s o l u t i o n , and s p e c i a l p o r t s
allow any d e s i r e d gas t o be bubbled i n t o t h e s o l u t i o n .
The mechanj.za1 t i m e r , mpgraph head and fulcrum may be a d j u s t e d i n
h e i g h t t o accommodate d i f f e r e n t s i z e d samples. The mechanical t i m e r i s on
an a d j u s t a b l e s l i d e which allows l o a d i n g and unloading of t h e weight a t any
p o i n t on t h e n u s c l e l e v e r .
The e n t i r e a p p a r a t u s i s mounted on 3/8 i n . aluminum p l a t e .
t o six m i t s can be mounted on t h e same p l a t e .
Four
Bzhler and F a l e s , (1966), designed a l e v e r system f o r t h e study of
t h e s e r i e s e l a s t i c component of muscle which appears t o be a p p l i c a b l e t o
s t u d i e s of e l a s t i c i t y changes during r i g o r .
Tension
The i n c r e a s e i n t e n s i o n caused by r i g o r s h o r t e n i n g i s a phenomenon
of t h e r i g o r p r o c e s s t h a t can be measured and appears t o be of p a r t i c u l a r
importance i n aging and t e n d e r n e s s s t u d i e s .
Jungk e t al (1967) d e s c r i b e an Isometer which w a s designed t o
measure t e n s i o n changes i n muscles h e l d i s o m e t r i c a l l y . T h i s a p p a r a t u s i s
unique i n t h a t muscle l e n g t h i s h e l d c o n s t a n t throughout t h e development of
r i g o r . It i s reasoned t h a t t h i s s i t u a t i o n more c l o s e l y approximates t h e
c o n d i t i o n of muscles i n t h e c a r c a s s undergoing r i g o r m o r t i s .
-
7
The isometer c o n s i s t s of a s i n g l e arm balance beam with a servo
mechanism c o n t r o l l i n g a counterweight p o s i t i o n t o keep t h e arm balanced. If
t h e muscle sample, which i s a t t a c h e d t o t h e balance beam t r i e s t o shorten,
t h e beam becomes unbalanced, a c t i v a t i n g t h e s e r v o system t o move t h e c o u n t e r weight t o balance t h e l e v e r a g a i n and r e t u r n t h e muscle t o i t s o r i g i n a l
l e n g t h . The movement of t h e counterweight i s t r a c e d by a r e c o r d e r , t h e r e b y
giving a t i m e tension relationship.
Length
Length changes can e a s i l y be measured by e x c i s i n g t h e muscle, plati n g it on a s u r f a c e t h a t a l l o w s t h e sample t o s h o r t e n f r e e l y and f o l l o w i n g
t h e change i n l e n g t h p e r u n i t of time (Jungk e t al 1 9 6 7 ) . The Myotron can
a l s o be used t o measure l e n g t h changes i n muscle h e l d under a c o n t r o l l e d
environment. The muscle sample would be l i g h t l y counterbalanced t o keep
t e n s i o n on t h e sample a t a minimum.
--
Cavagna et d, (1968) d e s c r i b e an ergometer f o r measuring t h e f o r c e
and change i n l e n g t h i n muscle d u r i n g e i t h e r s t r e t c h i n g or s h o r t e n i n g . T h i s
a p p a r a t u s would be a d a p t a b l e t o e i t h e r r i g o r s h o r t e n i n g measurements o r
s t u d i e s w i t h e l e c t r i c a l l y s t i m u l a t e d muscle.
Exc it ab ili t v C o n t r a c t il i t v
A s h o r t t i m e after death, s t r i a t e d muscle l o s e s i t s a b i l i t y t o
respond t o am e l e c t r i c a l s t i m u l a t i o n . T h i s l o s s of e x c i t a b i l i t y u s u a l l y
o c c u r s l o n g b e f o r e t h e completion of r i g o r m o r t i s ; however, i n p o r c i n e
12.
animals it can be demonstrated t h a t t h e e l e c t r i c a l response p r o p e r t i e s of
muscles t a k e n immediately a f t e r exsanguination are c o r r e l a t e d with t h e time
course of r i g o r m o r t i s ( F o r r e s t e t al 1966, 1 9 6 7 ) .
--
Muscles which go i n t o r i g o r r a p i d l y r e q u i r e a higher v o l t a g e
before g i v i n g a c o n t r a c t i l e response, t h e y give a weaker c o n t r a c t i l e response,
and t h e y w i l l not respond f o r a s long a d u r a t i o n a s muscles with a l o n g e r
t i m e source of r i g o r . T h i s method can be used t o p r e d i c t t h e time course of
r i g o r i n porcine muscle w i t h i n 1 0 min. of exsanguination.
Hardne s s
A s muscles s h o r t e n and t e n s i o n i n c r e a s e s t h e s u r f a c e of t h e m-uscle
becomes firm and ha,rd. The Mangold sclerometer d e s c r i b e d by Bendall (1960)
t a k e s advantage of t h i s phenomenon i n measuring r i g o r m o r t i s . Messtorf (1954)
also used t h i s p r i n c i p l e t o develop a blunt-ended p i s t o n t h a t i s pushed i n t o
t h e f l e s h by applying a c o n s t a n t p r e s s u r e t r a n s f e r r e d by way of a compression
s p r i n g . The depth p e n e t r a t i o n i s measured t o i n d i c a t e t h e degree of muscle
s t i f f e n i n g or hardening.
The p r e s s u r e e x e r t e d by t h e b l u n t p i s t o n does not b r i n g about any
change i n t h e mechanical c o n d i t i o n of t h e muscle of such a n a t u r e a s t o
i n f l u e n c e o r modify t h e t i m e course of r i g o r mortis, which i s an advantage
over many of t h e o t h e r methods.
Sarcomere l e n g t h
A s muscles s h o r t e n during t h e onset of r i g o r m o r t i s t h e l e n g t h of
t h e i n d i v i d u a l saxcomere u n i t s w i l l decrease. Stromer, Go11 and Roth (1967),
Stromer and Go11 (1967b) show electronmicrographs of bovine muscle i n v a r i o u s
s t a g e s of r i g o r development. Sarcomere l e n g t h can be determined by h i s t o l o g i c a l . methods u t i l i z i n g t h e phase c o n t r a s t microscope (Locker, 1959)
(Stromer and Goll 1967a). It i s p o s s i b l e although somewhat cumbersome t o
f o l l o w t h e development of r i g o r m o r t i s with t h e s e h i s t o l o g i c a l techniques.
They do have t h e important advantage of g i v i n g very s p e c i f i c information on
t h e c o n t r a c t i l e state of the muscle a t t h e v a r i o u s s t a g e s of r i g o r
de v e l opment
.
Mole c u l ar c o n f i g u r a t i o n
Very r e c e n t l y , low angle x - r a y d i f f r a c t i o n diagrams have been
obtained on s t r i a t e d muscle during c o n t r a c t i o n and during r i g o r (Huxley and
Brown 1 9 6 7 ) . Muscles i n r i g o r (when t h e c r o s s b r i d g e s a r e permanently
a t t a c h e d t o t h e t h i n f i l a m e n t s ) g i v e x-ray diagrams which d i f f e r q u i t e
c o n s i d e r a b l y from r e s t i n g or c o n t r a c t i n g muscle. The changes take place
l a r g e l y i n t h e myosin component and t h e r e s u l t s i n d i c a t e t h a t a re-organizat i o n of t h e h e l i c a l arrangement of t h e myosin c r o s s b r i d g e s may occur when
t h e y bind t o t h e s i t e s on t h e a c t i n f i l a m e n t s i n such a way a s t o maximize
t h e number of p o i n t s near r e g i s t r a t i o n . The a c t i n f i l a m e n t s appear t o behave
as r e l a t i v e l y i n v a x i a n t s t r u c t u r e s . However, s m a l l changes i n p i t c h may
occur.
The x-ray d i f f r a c t i o n technique o f f e r s some e x c i t i n g p o s s i b i l i t i e s
f o r g a i n i n g g r e a t e r i n s i g h t i n t o t h e s t r u c t u r e and f u n c t i o n of muscle, and
f u r t h e r p r e c i s e c h a r a c t e r i z a t i o n of t h e r i g o r process.
13.
Chemical Changes
M a n y chemical changes i n muscle are c l o s e l y a s s o c i a t e d with r i g o r
development. O f p a r t i c u l m importance and i n t e r e s t are t h e phosphate compounds, and many of t h e enzymes and i n t e r m e d i a t e s i n t h e g l y c o l y t i c pathways.
Following t h e s e chemical changes p o s t mortem g i v e s a good i n d i c a t i o n of t h e
r i g o r state of t h e muscle. Time does not permit t h e d i s c u s s i o n of t h e
i n s t r u m e n t a t i o n used i n t h e s e chemical and biochemical determinations.
summary
I n s t r u m e n t a t i o n i s a v a i l a b l e for t h e observation and q u a n t i t a t i o n
of n e a r l y e v e r y known p h y s i c a l change t h a t occurs i n muscle during t h e
development of rigor m o r t i s . Choice of parameters t o be measured depends
g r e a t l y upon t h e p a r t i c u l a r r e s e a r c h o b j e c t i v e . For example, e x t e n s i b i l i t y
changes a r e w e l l c o r r e l a t e d with p a l e s o f t exudative pork, sarcomere l e n g t h
and t e n s i o n a r e p a r t i c u l a r l y u s e f u l i n t e n d e r n e s s s t u d i e s , and x-ray d i f f r a c t i o n has been used f o r b a s i c s t u d i e s on s t r u c t u r a l changes during r i g o r .
I n most s t u d i e s involving post-mortem muscle an i n d i c a t i o n of t h e
p h y s i c a l s t a t e of t h e muscle would be u s e f u l . Whatever i n s t r u m e n t a t i o n i s
used, an a c c u r a t e d e s c r i p t i o n of t h e apparatus and t h e c o n d i t i o n s under which
t h e measurements were made i s e s s e n t i a l .
/-To
Recorder
Isotonic
Myoprog h
Transducer
Schematic drawing of t h e Myotron.
14.
Bahler, A. S., and J . T . F a l e s . A f l e x i b l e l e v e r system f o r q u a n t i t a k i v e
measurements of manmalian muscle dynamics
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e
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