Effect of high pressure oxygen exposure on red blood cells in splenectomized rat

Brooksby, G. A.

Chackerian, M. J.

Leon, H. A.

Staley, R. W.

English

NASA Technical Reports Server

I ' *  I .  - * -.c 8 
~ 
6 
BLOOD CHANGES 
.'., -4- 
H .  
IN ACUTELY SPLENECTQMIZEDJATS DURING PROLONGED HYPEROXIC EXPOSURE 
. Leon, G .  A .  Brooksby, M .  J .  Chackerian, R .  w.' 6 8  - 3 4 6 6 6 Biotechnology Division, Ames Research Center,  NASA 
I t  i s  genera l ly  accepted t h a t  changes i n  t h e  
SPLENECTOMIZED d S D RATS EXPOSED TO 600 mm Hg 0 2  c i r c u l a t i n g  red  c e l l  mass can be  e f f ec t ed  by O:, ac t -  
ing on t h e  rate of e ry th ropo ies i s .  
both t o  0 2  p a r t i a l  p ressures  g r e a t e r  than ambient 
which suppress RBC production and t o  hypoxia which 
s t imu la t e s  t h i s  process .  The p o s s i b i l i t y  t h a t  O2 
can a l t e r  t h e  r a t e  of  hemolysis has a l s o  been con- 
s ide red .  Thus, 0 2  a t  high pressure  has been shown 
t o  inc rease  hemolysis i n  v i vo  i n  tocopherol de f i -  
c i e n t  mice (1). But, as Kaplan poin ted  out  (2 )  , 
t h e  ques t ion  as t o  whether moderate increases  i n  
02 can inc rease  hemolysis i n  otherwise normal an i -  
mals o r  humans remains l a rge ly  unanswered. 
The sp leen  i s  a major s i t e  of RBC des t ruc t ion  
as well  as s to rage .  Considerable evidence sug- 
ges t s  t h a t  t h e  sp leen  can s e l e c t i v e l y  seques t e r  
t h e  o lde r  more f r a g i l e  R B C ' s .  Thus, it i s  prob- 
a b l e  t h a t  a sampling procedure t h a t  causes sp l en ic  
cont rac t ion  could r ap id ly  change both the  q u a l i t a -  
t i v e  and t h e  q u a n t i t a t i v e  na ture  of t h e  c i r c u l a t -  
ing  red  c e l l  mass. I n  t h i s  l a t t e r - r e g a r d ,  i t  i s  
well demonstrated t h a t  s p l e n i c  cont rac t ion  induced 
by epinephrine can inc rease  t h e  t o t a l  blood volume 
by 6-15% with a d i spropor t iona te ly  g r e a t e r  e f f e c t  
on the  hematocrit  due t o  t h e  concent ra t ion  of t h e  
discharge.  
I t  was f e l t  t h a t  any e f f e c t  of 0 2  on c i r c u l a t -  
ing  R B C ' s  would be more evident i n  splenectomized 
rats. The experiments repor ted  he re in ,  which a r e  
pre l iminary ,  show t h a t  with such rats,  cons i s t en t  
changes i n  blood, i n d i c a t i v e  of an increased  hemo- 
l y t i c  process ,  can b e  e f f e c t e d  by exposure t o  0 2  
a t  p a r t i a l  p ressures  below 1 atm. 
METHODS 
This app l i e s  
Male S-D r a t s  weighing 250 gm were splenec- 
I n  an i n i t i a l  tomized 4 days p r i o r  t o  exposure. 
s e r i e s  of  experiments, groups of  6 rats were 
exposed t o  600 mm Hg O 2  i n  ind iv idua l  chambers (3) 
f o r  0, 2 ,  4, 8 ,  14, and 28 days. These were com- 
pared  t o  an equal number of pa i r - f ed ,  splenecto- 
mized a i r - c o n t r o l  rats kept i n  metabolic cages.  
Hematocrit,  hemoglobin, methemoglobin, serum 
b i l i r u b i n ,  and osmotic f r a g i l i t y  curves were 
determined on both groups a t  t h e  s t a t e d  i n t e r v a l s .  
I n  another series, r a t s  s i m i l a r l y  t r e a t e d  were 
exposed t o  600, 450, 258, o r  190 mm Hg 02 f o r  14 
o r  28 days. 
tomized o r  sham-operated pa i r - f ed  con t ro l s .  
Hematocrit,  hemoglobin, and osmotic f r a g i l i t i e s  
were determined. 
and d i a l u r i c  ac id  hemolysis, a measure of an t iox i -  
dant  r e s e r v e s ,  were a l s o  measured. 
RESULTS 
These were compared t o  both splenec- 
RBC reduced g lu ta th ione  content 
.... i n e  resuits of t ne  600 mm i-ig 0, exposure 
study are i l l u s t r a t e d  i n  Figures 1 and 2 .  The 
hematocrit  i n  Figure 1 i s  s i g n i f i c a n t l y  depressed 
af ter  2 days ( 3 8  VS. 4 2 % ) .  This t rend  i s  s t i l l  
ev ident  a f t e r  28 days. 
t o t a l  Hb of  t h e  exposed rats suggest a r e l a t i v e  
RBC l o s s .  Methb and b i l i r u b i n  showed no s i g n i f i -  
can t  dev ia t ions  from those of t h e  cont ro l  r a t s .  
F igure  2 i l l u s t r a t e s  t h e  s i g n i f i c a n t  f r a g i l -  
Congruent changes i n  t h e  
/+-------- I 
HEMATOCRIT , 
I 
TOTAL MEAN f S E 
HEMOGLOBIN - 0 2  EXPOSED 
WIR FED CONTROL --- 
0 NORMAL, INTACT E : A , b M - ;  METHEMOGLOBIN 
s o  - 
E .4 r 
TOTAL SERUM 
0 2 4  E 14 2 8  
0 EXPOSURE, days 
Figure 1 
ERYTHROCYTE FRAGILITY OF SPLENECTOMIZED CT S.D. RATS 
EXPOSED TO 600 mm Hg 0, 
.40 percent SALINE 
100 _ _  --- - _ _ _  MEAN f S.E. - 02 EXPOSED I K j  -- 0 PAIR NO MAL, FED INTACT CONTROL 
80 
'J.' I 
60 
PERCENT 
HEMOLYSIS 
0 4 8 12 16 20 24 28 
02 EXPOSURE, days 
Figure 2 
-exposed rats show s i z a b l e  decreases i n  f rag i l -  0 2  
i t y  by t h e  e ighth  day. In  another s e r i e s ,  si;- 
n i f i c a n t  decreases were noted after only 3 days 
of exposure t o  600 mm Hg. A s  i n  t he  case of t h e  
hematocrits  and Hb content ,  t h e  changes tend t o  
p e r s i s t  f o r  a t  l e a s t  28 days. 
Osmotic f r a g i l i t y  was determined i n  s a l i n e  
concentrations from 0 .2% which caused 100% hemo- 
l y s i s  t o  0 .55% where l y s i s  was neg l ig ib l e .  How- 
ever ,  t h e  g r e a t e s t  decreases i n  f r a g i l i t y  
occurred i n  t h e  h ighe r  s a l i n e  concent ra t ions .  
The e s t ab l i shed  r e l a t ionsh ips  between 
osmotic f r a g i l i t y ,  volume, and age of RBC' s  (4) 
suggest t h a t  t h e  above changes i n  f r a g i l i t y  a r e  
due t o  a r e l a t i v e  lack of  o lde r ,  more f r a g i l e  
c e l l s  found i n  a normal population. 
it is 
decrease i n  r e t i cu locy te s  was seen only a f t e r  
14 days and was no longer evident a f t e r  28 days. 
In t h i s  
regard, interesting tfrat a significmt 
The hematocrit  (Fig.  3) was s i g n i f i c a n t l y  
A t  n e i t h e r  time 
suppressed by exposure t o  258 mm Hg a f t e r  2 weeks 
and t o  450 mm Hg f o r  4 weeks. 
i n t e r v a l  was t h e  hematocrit  depressed by exposure 
t o  190 mm Hg 0 2  which gives an a lveo la r  pO2 j u s t  
below normal. Hb content (Fig.  4) showed a s i m i -  
l a r  t rend ,  and was s i g n i f i c a n t l y  depressed a t  
https://ntrs.nasa.gov/search.jsp?R=19680025194 2020-03-23T23:10:58+00:00Z
*% . , #' 
d 
'* ,? HEMATOCRIT 
PERCENT 42 2 WEEKS 
RBC EXPOSURE 
38 
SPLENECTOMIZED 
0, EXPOSED 
SPLENECTOMIZED 
PAIR FED CONTROL 
- 
-- 
46 5 0 [  6 , / /I 
PERCENT 42 1 k\ 4 WEEKS 
RBC EXPOSURE 
b... 
3 4  ""LA 190 258 4 5 0  
02 PRESSURE, mm Hg 
Figure 3 
BLOOD HEMOGLOBIN CONTENT 
SPLENECTOMIZED 
02 EXPOSED 
SPLENECTOMIZED 
10 
l6 r PAIR FED CONTROL -- 
4 WEEKS 
EXPOSURE 
12 
190 258 4 5 0  
o2 PRESSURE, mm ~g 
10 L -~ I ' 
Figure 4 
both 258 and 450 mm Hg a f t e r  2 weeks and a t  
450 mm Hg a f t e r  4 weeks. 
were observed between t h e  190 mm Hg group and i t s  
con t ro l s .  
occurred i n  0.40% s a l i n e .  A t  both 258 and 450 mm 
Hg 02 osmotic f r a g i l i t y  was s i g n i f i c a n t l y  reduced 
after 2 weeks o f  exposure (Fig.  5 ) .  On t he  o the r  
hand, f r a g i l i t y  a t  a l l  t h ree  02 pressures  i s  
e s s e n t i a l l y  similar t o  t h a t  of t he  a i r  cont ro ls  
a f t e r  4 weeks o f  exposure. Thus, t he  f r a g i l i t y  
changes induced i n i t i a l l y  by exposure t o  02 a t  258 
o r  450 mm Hg appear s e l f - l i m i t i n g ;  i n  fact ,  pre-  
exposure f r a g i l i t i e s  appear t o  be  rees tab l i shed  
af ter  4 weeks d e s p i t e  continued exposure. 
Again, no d i f fe rences  
In  t h i s  series, the  g r e a t e s t  f r a g i l i t y  changes 
OSMOTIC FRAGILITY 
80 c 1 
70c t i  7' 2 WEEKS EXFOSUpE 
SPLENECTOMIZED 
02 EXPOSED 
- - SPLENECTOMIZED 
PAIR FED CONTROL 
Ii 
DISCUSSION 
The very na ture  of s p l e n i c  function'would 
suggest  t h a t  sequestered c e l l s  a r e  of an o lde r  pop- 
u l a t ion  d i s t r i b u t i o n  than t h e  c i r c u l a t i n g  blood; 
t he re fo re ,  s p l e n i c  cont rac t ion  would tend t o  
increase  the  proport ion of o l d e r  more f r a g i l e  c e l l s  
i n  t h e  c i r c u l a t i o n  and would inc rease  the  hemato- 
c r i t .  The occurrence of  t h i s  event j u s t  p r i o r  t o  
o r  during a sampling procedure could lead t o  an 
erroneous conclusion concerning t h e  e f f e c t s  of 
some previous condi t ion on t h e  blood. 
I t  i s  recognized t h a t  a decrease i n  hematocri t  
o r  Hb alone i s  not  s u f f i c i e n t  evidence t o  v e r i f y  a 
decrease i n  red c e l l  mass, and these  techniques 
were employed merely as ind ica to r s .  
The cons i s t en t  decreases  i n  f r a g i l i t y  ind ica t e  
s t rong ly  t h a t  exposure t o  a p02 as low as 258 mm Hg 
somehow s t imula tes  t he  removal of t he  more f r a g i l e  
c e l l s ,  f o r  i f  02 were merely t o  suppress hemopoi- 
e s i s ,  one would not  expect  t o  see  a decrease i n  t h e  
o lde r  more f r a g i l e  c e l l s .  On the  o the r  hand, i f  
peroxida t ive  hemolysis were occurr ing i n  the  face  
of continued 02 exposure, RBC's would be expected 
t o  show a g r e a t e r  f r a g i l i t y .  
evidence of  increased d i a l u r i c  ac id  hemolysis could 
b e  demonstrated even a f t e r  10 days of exposure a t  
In  t h i s  regard,  no 
600 IIUII Hg 02 .  
SUMMARY 
Exposing splenectomized rats t o  0, a t  pres-  
sures  from 600 t o  258 mm Hg cons i s t en t ly  changes 
hematocri t ,  t o t a l  Hb, and osmotic f r a g i l i t i e s  of 
RBC's, ind ica t ing  t h a t  t h e  c i r c u l a t i n g  red c e l l  
mass has been decreased by an ac t ive  removal of 
t h e  o lde r  o r  more f r a g i l e  c e l l s .  These changes 
have been observed wi th in  3 days bu t  appear 
s e l f - l i m i t i n g .  
REFERENCES 
1. Mengel, C .  E .  and Kann, H .  E . ,  Jr . :  J .  CZin. 
2 .  Kaplan, H T P . :  Aerospace Med. 2 : 6 7 6 ,  1967. 
3 .  Brooksby, G .  A . ,  Dennis, R .  L . ,  and S ta l ey ,  
Invest .  45:1150, 1966. 
R .  W . :  
on Hyperbaric Medicine, Natl . Acad. Sci  . , 
Washington, D . C . ,  1966, p .  208. 
4 .  Danon, D .  and Perk, K.: J .  Cellular Comp. 
Physioz. 59:117, 1962. 
Proc. Third IntemationaZ Conference 
- 
Note: 
* = p < 0.05 
** = p < 0.01 
*** = p < 0.001 
4 WEEKS EXPOSURE 
HEMOLYSIS 0.40% SALINE 
60 
190 258 4 5 0  
0 2  PRESSURE, mm Hg 
Figure 5 


Blood changes in acutely splenectomized rats during prolonged hyperoxic exposure

Abstract

Similar works

Full text

Available Versions

NASA Technical Reports Server