File Name: SLS1_16.DOC

EXPERIMENT SLS1 BSP16

SPINAL CORD: MORPHOLOGY AND HISTOCHEMISTRY

[PIs]{.underline}

[Russia:]{.underline}

I. B. Krasnov, Institute of Biomedical Problems, Moscow

[USA:]{.underline}

R. Edgerton, University of California, Los-Angeles

O. Lowry, Medical School, Washington University, Saint-Louis, Missouri

[Co-Investigators:]{.underline}

[Russia:]{.underline}

V. I. Drobyshev, Medical Institute, Voronezh

I. V. Polyakov, Medical Institute, Voronezh

[Introduction]{.underline}

Cytochemical and morphometric examinations of motoneurons of the
anterior horns of the lumbar and cervical enlargements of the spinal
cord of rats flown on Cosmos-605 \[5\], Cosmos-782 \[3\], Cosmos-936
\[1\], Cosmos-1887, and Cosmos-2044 \[7\] revealed changes suggestive of
a lowered activity of the nerve cells after 14-22-day exposure to
weightlessness. In order to assess the effect of a shorter-term exposure
to weightlessness on motoneurons of anterior horns, motoneurons as well
as capillaries of the anterior horns of the lumbar and cervical
enlargements of the spinal cord of rats flown for 9 days on SLS-1 were
investigated by means of quantitative cytochemical and histochemical
methods.

[Materials and Methods]{.underline}

Spinal cord at the level of the cervical enlargement (C2 - C4) and
lumbar enlargement (L1 - L3) of rats flown for 9 days on SLS-1 was
investigated. Rats formed the following groups, 5 animals each: flight
rats that were sacrificed 2 hours after recovery (R+0 group), flight
rats that were sacrificed 9 days after recovery (R+9 group), respective
vivarium controls (C+0 and C+9 groups). Spinal cord fragments were
placed into aluminum foil boats filled with 8% gelatin and frozen in
Freon-12 cooled to -150 oC in liquid nitrogen. Spinal cord fragments
were dissected and frozen in Freon-12 by a U.S. investigator at the
recovery site. Then the boats with frozen spinal cord samples were
shipped to NASA ARC where they were stored in liquid nitrogen and then
transported at -70 oC in stoppered Teflon vials filled with water ice
precooled to - 70 oC to the Institute of Biomedical Problems, Moscow.
Prior to cutting, spinal cord samples of R+0 and C+0 rats and R+9 and
C+9 rats were fixed pair-wise to prepare cryostat sections of the flight
and control animals of identical thickness on the same slide glass. The
sections were 12 μm thick. Using histochemical techniques, activities of
acetyl cholinesterase (ACE) \[6\], cytochrome oxidase (CO) \[8\] and
alkaline phosphatase (AP) were detected in cryostat sections. For AP,
α-naphthyl phosphate was used as a substrate and fast blue PP as an azo
component \[4\]. To arrest enzyme diffusion, the incubation medium was
supplemented with sucrose at a concentration of 155 mg/ml. The intensity
of histochemical reactions in the bodies of 40 motoneurons from each
animal was measured densitometrically at 480 μm for ACE and at 450 μm
for CO by means of a Luman I3 microscope equipped with a photometric
MEFP-1 unit with a 20-fold objective and a probe of 2 μm in diameter.
Enzyme activity was expressed as units of optical density. Blood
capillaries where AP

was detected were considered to be \"active capillaries\" that allowed
active metabolite transport across their walls. They were quantified
stereologically using a grid in the microscope ocular (ocular 10x,
objective 40x) and described as the number of capillaries in a 256 μm2
field of vision. \"Active capillaries\" were counted in 30 fields of
vision for each animal. The results obtained were processed using the
Student-Fisher test. Significant differences were determined by means of
statistical parametrical methods.

[Results and Discussion]{.underline}

Visual examination of the histochemical preparations did not indicate
any differences in the ACE distribution in motoneurons of the cervical
enlargement of spinal cord of the R+0 rats as compared to that of the
C+0 rats. However, the number of axonal endings, where ACE was detected,
on motoneuron body in the R+0 rats was lower than in the C+0 rats. This
can be ascribed to the flight-induced reduction in the level of
impulsation in cholinergic fibers that end on motoneuronal bodies.
Nevertheless, ACE activity in motoneuronal perikaryons of the R+0
animals remained unaltered (Table 1). The pattern of CO distribution in
motoneurons of the cervical enlargement of spinal cord of the R+0 rats
did not differ from that in the C+0 rats. This gives indirect evidence
that the structure and size of mitochondria in those nerve cells did not
change. Quantitative analysis of CO activity showed no changes in
motoneurons of the R+0 rats (Table 1). The number of \"active
capillaries\" in the anterior horns of spinal cord of the R+0 rats was
not different from that of the C+0 rats (Table 1). No signs of AP
diffusion were seen, pointing to the integrity of capillary endothelium.

Thus, no changes were observed in the cervical enlargement of spinal
cord of rats exposed to a 9-day space flight. However, an examination of
the cervical enlargement of spinal corn of Cosmos-2044 rats flown for 14
days \[7\] demonstrated a significant decrease in ACE in motoneurons and
a reduction in the number of \"active capillaries\"

in anterior horns. It can be postulated that 9 days in space were not
sufficient to reduce the function of motoneurons located in the anterior
horns of the cervical enlargement to

a detectable level.

Visual examination of histochemical preparations of the lumbar
enlargement of spinal cord of the R+0 rats did not reveal any
differences in the distribution of ACE activity when compared to the C+0
rats. The level of ACE activity in motoneurons of the R+0 rats was not
different from that in the C+0 rats either (Table 2). However, analysis
of the relationship between ACE activity and exposure time of the R+0
rats to the Earth\'s gravity showed that ACE activity in flight rat No.
21 which was exposed to the Earth\'s

gravity for 2 hours 15 minutes was 12% lower than in the C+0 rats,
whereas in flight rats Nos. 22 - 25 the parameter increased with
exposure time (Figure 1). Visual examination of CO distribution in
motoneurons demonstrated that the enzyme activity diminished uniformly
in the R+0 rats. This finding was confirmed by cytophotometric data: CO
activity declined significantly (by 16%, p \< 0.05) in motoneurons of
the R+0 rats when compared to the C+0 rats (Table 2). It is known that
CO activity in a neuron is an expression of oxidative metabolism which
provides a nerve cell with the energy required and can act as an
indicator of functional activity of the neuron. Therefore, a decline in
CO activity in motoneurons of the lumbar enlargement of spinal cord of
the R+0 rats reflects a decrease in the functional activity of these
neurons, probably, due to a reduced influx of afferent signals to them
in weightlessness. Activity and distribution of AP in the capillary
endothelium of anterior horns of the lumbar enlargement of spinal cord
of the R+0 rats did not differ from those in the C+0 rats (Table 2).

Histochemical examination of the cervical enlargement of spinal cord of
the R+9 rats did not identify any changes of ACE and CO activities in
motoneurons (Table 3). However, mention should be made of a significant
increase in the number of \"active capillaries\" in the anterior horns
of spinal cord of the R+9 rats. Taking into consideration the fact that
AP is responsible for active metabolite transport across the vascular
wall \[3\], this enhanced enzyme activity can be regarded as a
manifestation of a compensatory reaction stimulating metabolism in the
recovery period.

Histochemical analysis of structures of the anterior horns of the lumbar
enlargement of spinal cord of the R+9 rats did not show any qualitative
or quantitative differences in the distribution and level of ACE, CO or
AP activities between the R+9 and C+9 rats (Table 4).

Quantitative cytochemical analysis of ACE, CO and AP activities in the
anterior horns of the cervical and lumbar enlargements of spinal cord of
rats flown for 9 days on SLS-1 and then exposed to the Earth\'s gravity
for 2-3 hours or 9 days allows the conclusion that the activity of these
enzymes remained unchanged at the C2-C4 level and the activity of CO
decreased at the L1-L3 level. The latter finding suggests that as early
as 9 days after exposure to weightlessness the function of motoneurons
in the lumbar enlargement diminished. However, the fact that 9 days
after recovery CO activity in motoneurons of the lumbar enlargement
returned to the norm indicates that the changes were reversible and that
functional activity of motoneurons was easily restored. Comparison of
these observations with previously reported data about more distinct
changes in motoneurons of the anterior horns of spinal cord of rats
exposed to weightlessness for a longer period of time (14 - 22 days)
\[1, 3, 5, 7\] gives evidence that

the function of motoneurons continues to decline as the animal exposure
to weightlessness increases.

Increase in the number of \"active capillaries\" in the anterior horns
of the lumbar enlargement of spinal cord at R+9 day points to enhanced
transport of metabolites across capillaries and suggests the development
of compensatory processes that are aimed at stimulating metabolism in
spinal cord after recovery.

[Table 1]{.underline}

Histochemical analysis of motoneurons and capillaries in the cervical
enlargement of spinal cord of rats flown on SLS-1 for 9 days and exposed
to Earth\'s gravity for 2 hours (R+0 group)

  --------------------------- -------------------------------------------
  Parameter                   Rat group

  --------------------------- -------------------------------------------

  --------------------------- --------------------- ---------------------
                              R + 0                 C + 0

  --------------------------- --------------------- ---------------------

  --------------------------- --------------------- ---------------------
  ACE activity in             0.380 [+]{.underline} 0.390 [+]{.underline}
  motoneurons, optical        0.017                 0.027
  density units (M                                  
  [+]{.underline} m)                                

  --------------------------- --------------------- ---------------------

  --------------------------- --------------------- ---------------------
  CO activity in motoneurons, 0.460 [+]{.underline} 0.459 [+]{.underline}
  optical density units (M    0.031                 0.029
  [+]{.underline} m)                                

  --------------------------- --------------------- ---------------------

  --------------------------- --------------------- ---------------------
  \"Active capillaries\",     12.64 [+]{.underline} 11.88 [+]{.underline}
  number in the field of      0.62                  1.30
  vision (M [+]{.underline}                         
  m)                                                

  --------------------------- --------------------- ---------------------

[Table 2]{.underline}

Histochemical analysis of motoneurons and capillaries in the lumbar
enlargement of spinal cord of rats flown on SLS-1 for 9 days and exposed
to Earth\'s gravity for 2 hours (R+0 group)

  --------------------------- -------------------------------------------
  Parameter                   Rat group

  --------------------------- -------------------------------------------

  --------------------------- --------------------- ---------------------
                              R + 0                 C + 0

  --------------------------- --------------------- ---------------------

  --------------------------- --------------------- ---------------------
  ACE activity in             0.322 [+]{.underline} 0.305 [+]{.underline}
  motoneurons, optical        0.022                 0.026
  density units (M                                  
  [+]{.underline} m)                                

  --------------------------- --------------------- ---------------------

  --------------------------- --------------------- ---------------------
  CO activity in motoneurons, 0.350 [+]{.underline} 0.416 [+]{.underline}
  optical density units (M    0.014\*               0.022
  [+]{.underline} m)                                

  --------------------------- --------------------- ---------------------

  --------------------------- --------------------- ---------------------
  \"Active capillaries\",     12.28 [+]{.underline} 11.28 [+]{.underline}
  number in the field of      0.66                  0.61
  vision (M [+]{.underline}                         
  m)                                                

  --------------------------- --------------------- ---------------------

\* - p \< 0.005 as compared to the C+0 rats

[Table 3]{.underline}

Histochemical analysis of motoneurons and capillaries in the cervical
enlargement of spinal cord of rats flown on SLS-1 for 9 days and exposed
to Earth\'s gravity for 9 days (R+9 group)

  --------------------------- -------------------------------------------
  Parameter                   Rat group

  --------------------------- -------------------------------------------

  --------------------------- --------------------- ---------------------
                              R + 9                 C + 9

  --------------------------- --------------------- ---------------------

  --------------------------- --------------------- ---------------------
  ACE activity in             0.358 [+]{.underline} 0.370 [+]{.underline}
  motoneurons, optical        0.015                 0.017
  density units (M                                  
  [+]{.underline} m)                                

  --------------------------- --------------------- ---------------------

  --------------------------- --------------------- ---------------------
  CO activity in motoneurons, 0.467 [+]{.underline} 0.453 [+]{.underline}
  optical density units (M    0.010                 0.022
  [+]{.underline} m)                                

  --------------------------- --------------------- ---------------------

  --------------------------- --------------------- ---------------------
  \"Active capillaries\",     13.84 [+]{.underline} 11.42 [+]{.underline}
  number in the field of      0.59\*                0.50
  vision (M [+]{.underline}                         
  m)                                                

  --------------------------- --------------------- ---------------------

\* p \< 0.05 as compared to the C+9 rats

[Table 4]{.underline}

Histochemical analysis of motoneurons and capillaries in the lumbar
enlargement of spinal cord of rats flown on SLS-1 for 9 days and exposed
to Earth\'s gravity for 9 days (R+9 group)

  --------------------------- -------------------------------------------
  Parameter                   Rat group

  --------------------------- -------------------------------------------

  --------------------------- --------------------- ---------------------
                              R + 9                 C + 9

  --------------------------- --------------------- ---------------------

  --------------------------- --------------------- ---------------------
  ACE activity in             0.296 [+]{.underline} 0.298 [+]{.underline}
  motoneurons, optical        0.014                 0.013
  density units (M                                  
  [+]{.underline} m)                                

  --------------------------- --------------------- ---------------------

  --------------------------- --------------------- ---------------------
  CO activity in motoneurons, 0.416 [+]{.underline} 0.424 [+]{.underline}
  optical density units (M    0.018                 0.009
  [+]{.underline} m)                                

  --------------------------- --------------------- ---------------------

  --------------------------- --------------------- ---------------------
  \"Active capillaries\",     11.58 [+]{.underline} 10.60 [+]{.underline}
  number in the field of      0.60                  0.32
  vision (M [+]{.underline}                         
  m)                                                

  --------------------------- --------------------- ---------------------

![](media/image1.pct){width="6.805555555555555in"
height="7.416666666666667in"}

[References]{.underline}

1\. Gorbunova, A. V., Portugalov, V. V. The Effect of Artificial Gravity
in Space Flight on the Protein and RNA Content in Motoneurons of
Anterior Horns of Spinal Cord of Rats. Bull. Exper. Biol. Med., 1980,
vol. 90, No. 9, pp. 372-375 (in Russian).

2\. Motavkin, P. A., Lomakin, A. V., Chertok, V.M. Capillaries of the
Brain. Institute of Marine Biology, Far East Branch, USSR Academy of
Sciences, Vladivostok, 1983, 140 pp. (in Russian).

3\. Portugalov, V. V., Gorbunova, A. V., Petrova, N. V. Development of
Metabolic Changes in the Structures of the Spinal Reflex Arc of Rats
After Space Flight. Izv. AN SSSR, Ser. Biol., 1979, No. 1, pp. 18-27 (in
Russian).

4\. Burstone, M. S. Enzyme Histochemistry and Its Application in the
Study of Neoplasms. Acad. Press, 1962. New York, London.

5\. Gorbunova, A. V., Portugalov, V. V. Cytochemical Investigations of
Proteins and RNA in Spinal Motoneurons and Neurons of Spinal Ganglia of
the Rat After Space Flight. Aviat. Space Environ. Med., 1976, vol. 47,
No. 7, pp. 708-710.

6\. Karnovsky, M. J., Roots, L. A \"Direct-Coloring\" Thiocholine Method
for Cholinesterases. J. Histochem. Cytochem., 1964, vol. 12, No. 3, pp.
219-221.

7\. Krasnov, I. B., Polyakov, I. V., Drobyshev, V. I.
Neuron-Glia-Capillary System in Spinal Cord of Rats After 14 Day Space
Flight. The Physiologist, 1992, vol. 35, No. 1, Suppl., pp. 218-219.

8\. Wong-Riley, M. Changes in the Visual System of Monocularly Sutured
or Enucleated Cats Demonstrable with Cytochrome Oxidase Histochemistry.
Brain Res., 1979, vol. 171, No. 1, pp. 11-28.

[Summary]{.underline}

Quantitative cytochemical and histochemical analysis of acetyl
cholinesterase, cytochrome oxidase and alkaline phosphatase activity in
the anterior horns of the cervical and lumbar enlargements of spinal
cord of rats flown for 9 days on SLS-1 and then exposed to the Earth\'s
gravity for 2-3 hours or 9 days gave evidence that the activity of those
enzymes remained unchanged at the C2-C4 level and that the activity of
cytochrome oxidase decreased at the L1-L3 level. The latter finding
suggests that as early as 9 days after exposure to weightlessness the
function of motoneurons in the lumbar enlargement diminished. However,
the fact that 9 days after recovery cytochrome oxidase activity in
motoneurons of the lumbar enlargement returned to the norm indicates
that the changes were reversible and that functional activity of
motoneurons was easily restored. Increased number of \"active
capillaries\" in the anterior horns of the lumbar enlargement of spinal
cord at R+9 day points to enhanced transport of metabolites across
capillaries and suggests the development of compensatory processes that
stimulated metabolism in spinal cord after recovery.