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- STRIATAL CYTOMEGALIC NEURONS CONTAINING NITRIC OXIDE ARE
ASSOCIATED
WITH EXPERIMENTAL PERINATAL ASPHYXIA: IMPLICATION OF COLD
TREATMENT
- C. FABIAN LOIDL, FRANCISCO CAPANI, JUAN J. LOPEZ-COSTA, ESTER
LOPEZ, ASIA
SELVIN-TESTA, JORGE PECCI-SAAVEDRA
Instituto de Biología Celular y Neurociencias Eduardo De
Robertis, LANAIS-MIE, Facultad de Medicina, Universidad de Buenos
Aires.
Key words: hypothermia, NADPH-diaforasa, perinatal
asphyxia, striatum-
- Summary
-
- Neuropathological
mechanisms triggered by excitatory aminoacids are known to involve
nitric oxide (NO). Neurons containing NO are histochemically
reactive to nicotinamide adenine dinucleotide phosphate diaphorase
(NADPH-d), which labels NO synthase in CNS. Sprague-Dawley male
rats subjected to perinatal asphyxia (PA) at 37°C, and PA plus
15°C hypothermia were evaluated when 6 months old by NADPH-d
histochemical reaction. Computarized image analysis was used for
quantification of stained sections. NADPH-d neurons in striatum
from subsevere and severe PA showed a significant increment in
soma size and dendritic process length versus control and
hypothermic treated rats. Post-ischemic damage neurons are
therefore involved in NO changes induced by PA that may be
prevented by hypothermia treatment.
Resumen-
- Neuronas
estriatales citomegálicas que expresan óxido nítrico se asocian
con asfixia perinatal
experimental: implicancias del tratamiento con frio
El óxido nítrico (NO) se encuentra directa o indirectamente
relacionado con mecanismos neuropatológicos iniciados con la
liberación de aminoácidos excitatorios. Se pueden detectar
histoquímicamente las neuronas NO+ por su reacción a la
nicotinamida adenina dinucleótido fosfato diaforasa (NADPH-d),
cofactor específico para la enzima NO sintetasa en el SNC. Se
estudiaron con NADPH-d secciones de núcleo estriado de ratas
Sprague-Dawley de 6 meses de edad que fueron expuestas a un modelo
de asfixia perinatal (PA) durante varios períodos de tiempo tanto
a 37°C como a 15°C (tratamiento hipotérmi-co). Para comparar el
patrón de tinción de los diferentes grupos experimentales se
realizó un estudio de análisis de imágenes cuantitativo. Las
neuronas estriatales NADPH-d+ de ratas de 6 meses que fueron
expuestas a PA subsevera y severa mostraron un aumento
significativo en sus prolongaciones dendríticas y del tamaño del
soma en relación a los controles, a los animales tratados con
hipotermia y a los restantes grupos de PA a 37°C. Estos datos
indican que existen cambios crónicos en las neuronas que expresan
NO del estriado post-isquémico y que estas alteraciones inducidas
por PA pueden ser prevenidas con el tratamiento hipotérmico.
-
- Postal adress: Dr. Jorge Pecci Saavedra, Instituto de
Biología Celular y Neurociencias.
Eduardo De Robertis, Facultad de Medicina, Universidad de Buenos
Aires, Paraguay 2155, 1121 Buenos Aires, Argentina.
-
- Received: 10-I-1996 Accepted: 21-II-1996
Perinatal asphyxia (PA) remains a major complication in childbirth
and its frequency is still high in spite of progress in health
care. Affected newborns are prone to present neurological sequelae
in the short- and long-term, their severity depending on the
length of oxygen deprivation. Thus, PA may lead to attentional
deficit, hyperactivity, epilepsy, mental retardation, motor
disorders, cerebral palsy or even death1. The basic cause of
cerebral hypoxia in PA is pre- and intra-partum ischemia that
produces a global brain neurotransmission alteration involving the
nigrostriatal pathway, developing in adult rats as a long-term
increase in basal striatal dopamine (DA) levels2. Hypothermia has
been shown to prove critical for rat survival following PA
induction. Indeed, 100% survival was achieved up to 15-16 min of
asphyxia when PA was induced at body temperature (37°C), but
dropped to 20% after 20-21 min, to fall to zero after 22 min of
asphyxia. However, when PA was induced at a lower temperature
(15°C), litter survival reached 100% even up to 100 min
asphyxia2.
So far, the pathological mechanism triggered after 16 min of PA
leading to chronic neurotrans-mission alterations evidenced in
monoamines and GABA2, 3 is poorly understood but may well involve
the participation of excitatory aminoacids, nitric oxide and free
radicals.
Nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d)
is commonly employed as a histochemical marker of NO synthesis in
neurons4 and it was the fact that NO had been described to enhance
DA release from striatum5 that encouraged us to evaluate NADPH-d
distribution.
As striatum is severely affected by PA, we attempted to delineate
the pattern of NADPH-d labelling in 6-month-old rat sections after
various PA periods, either with or without hypothermia treatment.
Sprague-Dawley rats on the last day of gestation were
anaesthetised with ether and hysterectomized, their gestational
age being determined by palpation. The entire uterus containing
the fetuses was taken out, the uterine horns were detached and
placed in a water bath at 37°C for 5 or 10 min (both regarded as
slight PA), 15 (moderate PA), 19 (subsevere PA) or over 20 (severe
PA)2, or else in a bath at 15°C for 20 or 100 min.
Cesarean-delivered control and asphyctic pups were obtained from
the same mother. Following induction of asphyxia, the uterine
horns were rapidly opened and pups removed and stimulated to
breathe on a heating pad by cleaning up the delivery fluid and by
tactile stimulation with medical wipes. The umbilical cord was
ligated and animals left to recover for around 1h before given to
surrogate mothers which had delivered normally 24 hs before the
experiment, mixing their normal litters with marked asphyctic and
caesarean-delivered control pups.
On becoming adults at six months of age (N = 4-5 animals/group),
they were anaesthetized with sodium pentobarbital (60 mg kg-1) and
perfused through the aortic artery with 0.9% NaCl solution
followed by 300ml of 4% paraformaldehyde in 0.1M phosphate buffer.
Brains were removed and postfixed in the same solution during 2hs,
then immersed overnight in a solution containing 20% sucrose in
0.1M phosphate buffer. Sections 40 µm thick were cut on an Oxford
vibratome and mounted on gelatin-coated glass slides, then
processed by the NADPH-d histochemical method. Briefly, sections
were incubated for 1h at 37°C in a solution containing 0.1%
b-NADPH and 0.02% nitroblue tetrazolium diluted in 0.1M phospate
buffer with 0.3% Triton X-100 (all reagents purchased from Sigma,
U.S.A.). Sections were mounted in PBS/glycerol (1: 3), then
observed and photographed with a Zeiss Axiophot microscope. Mean
number and cell perimeter of NADPH-d+ cells per random field from
10 striatal sections belonging to each group were calculated using
a KONTRON/VIDAS image analyzer, quantifying a total of 10 cells
per section. Sections were observed with a 40X objective, and
images digitized using an Axiophot Zeiss microscope linked to the
computer by a SONY video camera. Differences between groups were
compared using analysis of variance (ANOVA), taking p<0.05 as
significant. Results are given as means ± SD. Without exception,
NADPH-d staining in striatum transverse sections from 6-mont-old
rats showed uniformly distributed medium-sized neurons. Subsevere
and severe PA groups disclosed so-called neuronal cytomegaly6,
together with an evident increase in tortuous dendritic
arborizations compared to control, remaining PA groups at 37°C
and those subjected to 20 and 100 minutes PA at 15°C.
In rats exposed to subsevere and severe PA, striatal cell
perimeter disclosed highly significant cytomegaly (p<0.001)
versus control, slight and moderate PA at 37°C and PA at 15°C
groups during 20 or 100 minutes (Fig. 1 and Table 1). Mean
striatal cell count per field with a 40X objective was 5 ± 1,
lacking significant intergroup differences. In agreement with
reports describing NADPH-d+ striatal cells as interneurons
containing somatos-tatine and neuropeptide Y7, in adult rat such
neurons were of medium size and non-spiny type. Due to their lack
of NMDA receptors7 and high concentration of manganese superoxide
dismutase8, these cells were regarded to be resistant to
excitatory aminoacid toxicity and to free radicals toxicity
respectively. Nevertheless, the broader implications of NO as a
neurotoxic or neuropro-tective modulator remain a subject of
controversy. Several neurological disorders are liable to induce
changes in NADPH-d stained neurons. To illustrate, in Huntington's
disease there is an increase in striatal NADPH-d+ cell
measurements concomitantly with cell and fiber sparing, perhaps
due to loss of striatal spiny neurons7. A recent report on human
Parkinson's and Alzheimer's diseases has documented a relative
NADPH-d+ cell sparing in striatal neurons displaying shrunken and
foreshortened dendritic processes9. In the present study, an
experimental model was specially designed to evaluate PA effects
in adult animals2, 3. NADPH-d technique disclosed positive cells
of the same type, homogeneously distributed in asphyctic and
control rat striatum. However, striatal NADPH-d+ neurons from rats
subjected to subsevere or severe PA proved highly cyto-megalic
compared with control and hypothermia-treated animals, indicating
that the pathological mechanism involved in PA is dissimilar to
that in Alzheimer's, Parkinson's and Huntington's diseases.
Alterations found in rat striatal NADPH-d+ neurons closely
resembled those reported by Mischel et al6 who documented cortical
neuronal cytomegaly in pediatric epilepsy. As NO induces DA
release, which may be blocked by the NO synthase inhibitor
L-Me-Arg5, the higher basal level of striatal DA after an episode
of severe PA2 seems to agree with the number of NO-containing
hypertrophic striatal neurons.
Besides, NO plays a major role in regulating blood flow by
inducing relaxation of the vascular smooth muscle10, and most
likely protects against ischemia by enhancing oxygen supply. The
ability to stimulate DA release, considered toxic in striatum
after ischemia11, together with its potent vasoconstrictor effect
may worsen cell damage. However, since DA release is inhibited by
hypothermia12, its protective mechanism is attributable to
inhibition of the excessive release of striatal DA indirectly
stimulated by NO.
In spite of the therapeutic application of hypothermia to reduce
the severity of ischemic cerebral damage described in several
studies in rats and gerbils22, data on PA is mostly limited to the
1950s and 1960s, when cold therapy plus positive pressure
ventilation was introduced to resuscitate severely asphyxiated
human neonates, previously regarded as incapable or recovery, with
and excellent outcome evidenced by a rapid increase in Apgar
scores13, 14. Metabolic demands decrease when body temperature is
lowered15 so that toxic effects are weakened and survival to
asphyxia ensured without permanent brain lesions. However, the
functional consequences arising from striatal NO-containing
cytomegalic neurons after an episode of subsevere and severe PA
are still obscure, so that further studies on cold therapy in the
treatment of severely asphyctic babies are essential. In
conclusion, our findings demonstrate that NADPH-d reactivity is
enhanced in striatal neurons containing NO secondary to chronic
PA-induced ischemia due to a subsevere or severe lack of oxygen
and that permanent brain damage may be prevented by hypothermia
treatment.
Acnowledgements: We would like to express our gratitude for
the excellent technical assistance of Mrs Emérita Jorge Vilela de
Banchieri. This work was supported by grants from the University
of Buenos Aires and CONICET (Consejo Nacional de Investigaciones
Científicas y Técnicas).
-
- References
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Am J Obstet Gynecol 1971; 110: 1125-33.-
- Fig. 1.- Measurement of striatal NADPH-d+ cell perimeter, in
6-month-old rats subjected to different periods of PA at 37°C or
15°C. Each value represents mean ± SD (vertical lines) of
determinations made from n = 100 cells from each group. Asterisk
indicates that NADPH-d+ in these two groups was significantly
different (p<0.001) from the remainder. Statistical analysis
was performed by ANOVA test. There were no significant differences
between subsevere and severe PA.
- TABLE 1.- Cell perimeter or rat striatal neurons following
various periods of newborn
asphyxia
Time of asphyxia Perimeter
(minutes) (µm)
Mean SD
- Zero (control) 37°C 67.10 ± 28.11
5-6 37°C 77.66 ± 20.55
10-11 37°C 55.30 ± 16.82
15-16 37°C 53.79 ± 16.82
19-20 37°C 153.28 ± 81.12*
20-21 37°C 145.56 ± 73.57*
20-21 15°C 64.47 ± 29.64
100-101 15°C 54.01 ± 28.36
- · Significantly different from the remaining groups
(P<0.001) but not between them.
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