Vol. 281, Issue 4, H1476-H1480, October 2001
Cerebral arteriolar structure and function in pinealectomized
rats
Olivier
Régrigny1,
François
Dupuis1,
Jeffrey
Atkinson1,
Patrick
Limiñana1,
Elizabeth
Scalbert2,
Philippe
Delagrange2, and
Jean-Marc
Chillon1
1 Cardiovascular Research Group, Faculté de Pharmacie
de l'Université Henri Poincaré-Nancy I, 54000 Nancy; and
2 Institut de Recherches Internationales Servier, 92415 Courbevoie Cedex, France
 |
ABSTRACT |
We examined cerebral arteriolar structure and autoregulation of
cerebral blood flow (CBF) in control (n = 8),
sham-operated (n = 8), pinealectomized
(n = 10), and pinealectomized plus melatonin-treated (0.51 ± 0.01 mg · kg
1 · day1 in drinking
water, n = 9) young Wistar rats. The lower limit of CBF
autoregulation (LLCBF) was determined by measurement of CBF (in
arbitrary units, laser Doppler) during stepwise hypotensive hemorrhage;
the arteriolar internal diameter (ID; in µm, cranial window) was also
measured. Measurements of ID were repeated during a second stepwise
hypotension after smooth muscle cell deactivation (67 mmol/l EDTA). The
cross-sectional area (CSA) was measured by histometry. CSA and
EDTA-induced vasodilatation decreased after pinealectomy (517 ± 21 vs. 819 ± 40 µm2 in sham and 829 ± 55 µm2 in control, P < 0.05, and 81 ± 4 vs. 102 ± 5 µm in sham and 104 ± 4 µm in control,
P < 0.05, respectively) and were restored by melatonin
(924 ± 39 µm2 and 102 ± 5 µm,
respectively). These results suggest that melatonin deprival makes the
arteriolar wall thinner and stiffer. However, these changes had little
effect on LLCBF. In conclusion, pinealectomy of young rats induces
atrophy and decreases distensibility of the cerebral arteriolar wall;
these effects are prevented by melatonin. They do not modify LLCBF.
melatonin; autoregulation; atrophy; maximal vasodilatation
| |
INTRODUCTION |
WE RECENTLY REPORTED
that melatonin vasoconstricts cerebral arterioles via activation of
either MT1 or MT2 G protein-linked membrane
receptors (13). Vascular smooth muscle cell membrane receptors are also coupled to intracellular pathways (11)
leading to cellular hypertrophy (6). We thus speculated
that melatonin may also be involved in the control of vascular wall
thickness and that melatonin deprival (produced by pinealectomy) would
lead to atrophy of the vascular wall. Furthermore, because atrophy of
the vascular wall is associated with a decrease in passive distensibility (8), we hypothesized that melatonin
deprival would impair the distensibility of the cerebral arterioles and that this would impair cerebral blood flow (CBF) autoregulation.
The goal of this study was, therefore, to examine the effects of
melatonin deprival after pinealectomy of young normotensive rats on
cerebral arteriolar structure and function and on the lower limit of
CBF autoregulation. To evaluate whether the changes produced by
pinealectomy were solely due to melatonin deprival, we also studied
pinealectomized rats treated for 1 mo with melatonin.
| |
METHODS |
Animals and operative procedures.
Experiments were performed on male Wistar rats (Ico: WI, IOPS AF/Han,
Iffa-Credo; l'Arbresle, France; 378 ± 11 g body wt). At 8 wk of age, rats were divided into four groups as follows: control
(n = 8), sham-operated (n = 8),
pinealectomized (n = 10), and pinealectomized plus
melatonin-treated rats (6 mg/l in drinking water, n = 9). The dose of melatonin used in the present experiment was chosen on
the basis of a previous report (12) showing that treatment
with melatonin (4 mg/l) in drinking water produces a nocturnal plasma
melatonin level in 10-mo-old rats 15 times higher than that in 4-mo-old
rats. We decided to treat our rats with the slightly higher dose of 6 mg/l melatonin in drinking water supposing that such a dose would
produce plasma melatonin levels in pinealectomized rats at least 15 times higher than that in untreated pinealectomized rats. The average
daily intake of melatonin was 0.51 ± 0.02 mg · kg1 · day1.
The animals were pinealectomized under pentobarbital sodium anesthesia
(60 mg/kg). Briefly, the head was placed in a stereotaxic device, the skull was exposed, and a hole (5 mm in diameter) was drilled above the lambda. The superior sagittal venous sinus was incised, and the pineal gland, located underneath the sinus, was removed by aspiration. The skin was then sutured, and the rats received
a single injection of penicillin (120,000 IU/kg im). It has been
previously reported that pinealectomy significantly reduces serum
melatonin (17) and urinary 6-sulfatoxymelatonin (5) levels in rats. Because destruction of the superior
sagittal venous sinus may disrupt measurement of CBF, sham-operated
animals were submitted to the same procedure except that the pineal
gland was not removed.
Melatonin was prepared three times a week by dissolving the drug (12 mg) in ethanol (1 ml, 100% vol/vol). This solution was then diluted
with demineralized water to a final concentration of 6 mg/l
(concentration of ethanol, 0.05% vol/vol). A preliminary study showed
that melatonin is stable in such a solution for up to 3 days
(unpublished results). Other groups received solvent. Fluid consumption
was determined three times, and body weight was determined once a week.
Fluid consumption was similar in all groups (results not shown).
Animals were housed at 24°C, exposed to 12 h of light (lights on
at 6 AM and off at 6 PM), and allowed free access to food and fluid.
Experiments were performed in accordance with the guidelines of the
French Ministry of Agriculture (Paris, France) (permits 54-4 and 03575).
After 1 mo of treatment, we evaluated CBF autoregulation and the
structure and function of cerebral arterioles. Animals were anesthetized with pentobarbital sodium (60 mg/kg ip) at 9 AM, and a
polyethylene cannula (Merck Biotrol; Chennevieres, France) was
introduced into the left femoral artery; the cannula was connected to a
low-volume strain-gauge transducer (Baxter, Bentley Laboratories) for
measurement of blood pressure and heart rate. A second cannula was
introduced into the right femoral artery for blood withdrawal and
measurement of arterial blood gases at baseline and during hypotension.
A silicone catheter (Sigma Medical; Nanterre, France) introduced into a
femoral vein was connected to a pump (Bioblock Scientific; Paris,
France) for infusion of pentobarbital sodium (0.25 ml/h, 20 mg · kg1 · h1) to maintain
anesthesia throughout the experiment. Animals were intubated and
mechanically ventilated with room air (60 strokes/min; tidal volume,
2.3 ml) to maintain blood gases (pH, PCO2, and
PO2; blood gas analyzer 238, Ciba Corning;
Cergy Pontoise, France) in the physiological range. Paralysis of
skeletal muscles was obtained with gallamine triethiodide (20 mg/kg iv)
repeated every hour. Because the animals were paralyzed, the depth of
anesthesia was periodically evaluated by applying pressure to the tail
and observing changes in heart rate and blood pressure. Rectal
temperature was maintained at 37-38°C with a heating pad.
Measurement of arteriolar diameter.
We measured the internal diameter of first-order arterioles of the
cerebrum (9) through an open skull preparation (3, 4). The head was placed in an adjustable head holder, and a 1-cm
skin incision was made to expose the skull. A dam of dental acrylic was
constructed around the exposed skull, and ports were placed for inflow
and outflow of artificial cerebrospinal fluid (CSF). Craniotomy was
performed over the left parietal cortex, and the dura was incised to
expose cerebral vessels. Subarachnoid hemorrhage was not observed after
the craniotomy. The exposed brain was continuously suffused with
artificial CSF, warmed to 37-38°C, and equilibrated with a gas
mixture of 5% CO2-95% N2. The composition of
the CSF was (in mmol/l) 3.0 KCl, 0.6 MgCl2, 1.5 CaCl2, 131.9 NaCl, 24.6 NaHCO3, 6.7 urea, and
3.7 glucose (3, 4).
Arteriolar diameter was monitored through a microscope (Stemi 200-C,
Carl Zeiss; Jena, Germany) connected to a closed-circuit video system
with a final magnification of ×400. Images were digitized using a
video frame grabber, and diameter was measured using image analysis
software (Saisam, Microvision Instruments; Evry, France). The precision
of this system is 0.5 µm.
Measurement of CBF.
Relative changes in CBF were measured by laser Doppler flowmetry using
a BLF 21 system (Transonic Systems; Ithaca, NY) equipped with a
1.2-mm-diameter needle probe (7). The probe was placed in
the CSF of the cranial window. CBF was expressed as arbitrary units
(au) or as percent (from baseline) change in CBF (during stepwise hypotension).
Experimental protocol.
Thirty minutes after completion of surgery, cerebral arteriolar
diameter was measured at baseline. Stepwise hypotension (10 mmHg/step)
down to a systemic mean arterial pressure of 20-30 mmHg was
induced by controlled withdrawal of blood. At each step, systemic
pressure, arteriolar diameter, CBF, and blood gases were measured 1 min
after the fall in blood pressure. After the final step, blood was
reinjected to restore blood pressure.
Vascular smooth muscle was then deactivated by suffusion of cerebral
vessels with artificial CSF containing EDTA (67 mmol/l) (2), and the maximal vasodilator response was measured.
Systemic mean arterial pressure-diameter relationships were obtained in deactivated cerebral arterioles between systemic mean arterial pressures of 110 and 20 mmHg using hemorrhage to reduce pressure in
steps of 10 mmHg. At each pressure step, arteriolar diameter reached a
steady state within 15 s, and internal diameter was measured
30 s later. After the final step, blood was reinjected to restore
blood pressure. Arterioles maximally dilated by EDTA were then fixed in
vivo at prehemorrhage pressure by suffusion with glutaraldehyde (2.25%
vol/vol in 0.10 mol/l of cacodylate buffer). Arterioles were considered
to be adequately fixed when blood flow ceased. Lack of blood flow in
fixed arterioles was consecutive to fixation of red blood cells and not
to fixation-induced contraction of the artery. The animals were
euthanized with a pentobarbital sodium overdose (250 mg/kg), and
arteriolar segments used for pressure-diameter measurements were
removed and processed for paraffin embedding and light microscopy.
The cross-sectional area (CSA) of the arteriolar wall was determined on
7-µm sections using the video image analyzing system described above.
Luminal and total (lumen plus vessel wall) CSA were measured by tracing
the luminal and outer edges of the vessel wall. Wall CSA was calculated
by subtraction of luminal from total CSA.
Calculations.
Data for cerebral arteriolar diameter are reported as absolute values
(after EDTA) or as differences from baseline (during hypotension-induced vasodilatation). Data for CBF are reported as the
percent change from baseline. For each group, CBF (percent change from
baseline) and arterial pressure values were presented in the form used
by Barry et al. (1). Values were pooled and grouped by
categories over mean arterial pressure ranges of 10 mmHg. One-way ANOVA
within these different mean arterial pressure ranges was performed for
each treatment group. The lower limit of CBF autoregulation was defined
as the lower limit of the lowest mean arterial blood pressure range in
which CBF was not significantly less than baseline CBF. The security
margin (in %), which indicates the degree to which mean arterial
pressure may fall before CBF starts to decrease, was defined as
follows: [(baseline mean arterial blood pressure 
lower limit
of cerebral blood flow autoregulation)/(baseline mean arterial blood
pressure)] × 100 (10).
Substances used.
Gallamine triethiodide and melatonin were purchased from Sigma (St.
Louis, MO). Nitrogen and carbon dioxide were purchased from Air Liquide
(Nancy, France). Pentobarbital sodium was purchased from Sanofi
Santé Animale (Libourne, France). KCl, MgCl2,
CaCl2, NaCl, NaHCO3, urea, and glucose were
purchased from Merck (Darmstadt, Germany).
Statistical analysis.
Results are expressed as means ± SE. The experimental protocol
was designed for the use of a one-way ANOVA with the variable "treatments" (sham, pinealectomized, and pinealectomized plus melatonin). Significant differences between means were determined using
the Bonferroni test. ANOVA for hypotensive hemorrhage data is described
above. The probability level chosen was P 
0.05.
| |
RESULTS |
Baseline values.
Body weight was similar in the various groups at the end of the
experiment (Table 1). Cerebral arteriolar
internal diameter before deactivation with EDTA was not influenced by
pinealectomy, whereas CSA was decreased by pinealectomy and restored to
values similar to those of sham-operated or control rats after
treatment with melatonin (Table 1). Heart rate, blood pressure, CBF,
pH, and blood gases were similar in all groups of rats (Table 1).
Vascular mechanics.
After deactivation of cerebral arterioles with EDTA, internal diameters
were significantly less in pinealectomized rats than in sham-operated
rats at systemic mean pressures between 110 and 40 mmHg (Fig.
1B). Treatment with melatonin
restored the EDTA-induced dilatation in internal diameter in
pinealectomized rats (Fig. 1B). Internal diameters measured
after fixation of cerebral arterioles were significantly less than
internal diameters obtained in vivo in control, sham-operated, and
pinealectomized rats treated with melatonin (Table 1).
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Fig. 1.
Systemic mean arterial pressure-internal diameter
relationship in arterioles before (A) and during
(B) maximal dilatation with EDTA (67 mmol/l) in control
(n = 8), sham-operated (n = 8), and
pinealectomized rats that were untreated (n = 10) or
treated with melatonin (n = 9). Values are means ± SE. *P 0.05 vs. sham.
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Lower limit of CBF autoregulation, security margin, and
hypotension-induced dilatation of cerebral arterioles.
After hypotensive hemorrhage in control and pinealectomized rats, CBF
remained constant until the pressure range of 40-49 mmHg and then
significantly decreased; the lower limit of CBF autoregulation was 40 mmHg, and the security margin was 62 and 61%, respectively (Table 1
and Fig. 2). After hypotensive hemorrhage in sham-operated and melatonin-treated rats, CBF remained constant until the pressure range of 30-39 mmHg and then significantly decreased; the lower limit of CBF autoregulation was 30 mmHg, and the
security margin was 71% (Table 1 and Fig. 2).
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Fig. 2.
Cerebral blood flow (CBF) autoregulation during stepwise
hypotension in control (n = 8), sham-operated
(n = 8), and pinealectomized rats that were untreated
(n = 10) or treated with melatonin (n = 9). CBF values (percent baseline, ±SE) are grouped by mean arterial
blood pressure ranges of 10 mmHg (20-29 to 100-109 mmHg)
(1, 15).  P 0.05 vs. baseline values in
same group.
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In sham, pinealectomized, and melatonin-treated rats, cerebral
arterioles dilated significantly at pressures <70 mmHg. In control
rats, cerebral arterioles dilated at pressures <60 mmHg. Active
dilatation was slightly decreased in pinealectomized rats compared with
sham-operated rats or pinealectomized rats treated with melatonin (Fig.
3). Maximal dilatation (20.8 ± 2.6 µm in control, 20.0 ± 1.4 µm in sham-operated, 15.0 ± 1.2 µm in pinealectomized, and 22.0 ± 2.0 µm in
pinealectomized and melatonin-treated rats) was observed at 30-39
mmHg (Fig. 3). Systemic mean arterial pressure-internal diameter
relationships in cerebral arterioles with active tone were not
significantly different between the various groups (Fig. 1A).
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Fig. 3.
Variation in internal diameter at systemic arterial mean
pressure steps between 110 and 20 mmHg in arterioles during hypotensive
hemorrhage before EDTA in control (n = 8),
sham-operated (n = 8), and pinealectomized rats that
were untreated (n = 10) or treated with melatonin
(n = 9). Values are means ± SE. P 0.05 vs. baseline values in same group.
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| |
DISCUSSION |
The present study shows that pinealectomy reduces wall thickness
and EDTA-induced maximal vasodilatation without changing baseline
internal diameter of cerebral arterioles. Treatment with melatonin
restores normal wall thickness and maximal vasodilatation of cerebral
arterioles. Despite changes in structure and mechanics of cerebral
arterioles in pinealectomized rats, the consequences on the
hypotension-induced vasodilatation and lower limit of CBF autoregulation are minor.
Effects of melatonin on cerebral arteriolar wall structure and
mechanics.
Melatonin deprival for 1 mo reduced wall thickness; this was prevented
by melatonin treatment. This result confirms our hypothesis that
melatonin has a trophic effect on the cerebral arteriolar wall and
contributes to the maintenance of cerebral arteriolar wall mass. It has
previously been reported that cerebral arterioles undergo atrophy of
the vessel wall during aging (8). The mechanisms that
contribute to atrophy of cerebral arterioles during aging are not
clear. Because melatonin production decreases with age (14), this may be an element of the age-associated
reduction in wall thickness of cerebral arterioles. In old rats,
atrophy of the cerebral arteriolar vessel wall is associated with a
decrease in passive distensibility after a reduction in the more
distensible components of the vessel wall (elastin, smooth muscle, and
endothelial cells) (8). In the present study, we did not
calculate distensibility parameters because we measured systemic
pressure and not the pressure in cerebral arterioles. However, the
systemic mean pressure-internal diameter relationships obtained in
cerebral arterioles after EDTA (Fig. 1B) show that maximal
vasodilatation was impaired in cerebral arterioles of pinealectomized
rats at higher pressure steps. There are at least two explanations for
this result. First, because we did not measured pial arteriolar mean
pressure but used systemic mean pressure, we cannot rule out the
possibility that pial arteriolar mean pressure was different in
pinealectomized rats despite similar levels of systemic mean pressure.
Second, the pressure-diameter relationship may reflect a decrease in
the passive distensibility of the cerebral arteriolar wall. A decrease
in distensibility of cerebral arterioles after pinealectomy may explain
why the internal diameter measured by histometry in pinealectomized
rats was similar to internal diameter measured in vivo, whereas
internal diameter measured by histometry in control, sham-operated, and melatonin-treated rats was significantly smaller than internal diameter
measured in vivo.
Effects of melatonin on the lower limit of CBF autoregulation.
Despite the differences in arteriolar wall structure and maximal
vasodilatation after melatonin deprival, consequences on the lower
limit of CBF autoregulation were minor (Fig. 2). This result may be
explained by the fact that the hypotension-induced vasodilatation of
cerebral arterioles was only slightly decreased in pinealectomized
compared with sham-operated or control rats (Fig. 3). This result is in
apparent contradiction with the fact that maximal vasodilatation is
impaired in pinealectomized rats. We have to remain cautious, however,
because, to our knowledge, there is no direct evidence that a decrease
in the response to a "maximal" vasodilator stimulus such as EDTA is
associated with a decrease in vasodilatation to a "submaximal"
stimulus such as hypotension. Furthermore, we cannot rule out the
possibility that melatonin depletion affects other factors that might
modify the response of vessels to hemorrhage, such as adrenergic
innervation, for example. Finally, in the present experiment, we used
laser Doppler measurements and not actual blood flow to the whole
brain. Thus total blood flow may be decreased in melatonin-deficient animals even if CBF autoregulation appears preserved.
Conclusion and implications.
Pinealectomy of young normotensive rats induces atrophy of the cerebral
arteriolar wall associated with a decrease in passive distensibility.
Because these structural and mechanical alterations were reversed by
melatonin treatment, they were probably consecutive to melatonin
deprival. However, the structural and mechanical alterations had little
effect on the lower limit of CBF autoregulation in young rats, perhaps
following compensatory mechanisms.
Melatonin deprival in young normotensive rats induces changes in the
structure and mechanics of cerebral arterioles similar to those
observed in old rats (8) in which the lower limit of CBF
autoregulation increases (10). This, together with an increase in blood pressure variability (18), may
contribute to vascular dementia in aging (15, 16). Because
melatonin production decreases with age (14), this may be
an important element of the age-associated alteration in structure and
mechanics of cerebral arterioles and may play an important role in the
etiology of vascular dementia. This hypothesis remains to be tested.
| |
ACKNOWLEDGEMENTS |
This study was funded by French Ministry of Education, Research and
Technology (Paris, France) Grant EA3116 and by the Regional Development
Committee (Metz, France), the Greater Nancy Urban Council (Nancy,
France), Henri Poincaré University (Nancy, France), and the
Institut de Recherches Internationales Servier (Courbevoie, France).
| |
FOOTNOTES |
Address for reprint requests and other correspondence: J. Atkinson, Cardiovascular Research Group, EA 3116, Faculté de
Pharmacie de l'Université Henri Poincaré-Nancy I, 5 rue
Albert Lebrun, 54000 Nancy, France (E-mail:
atkinson{at}pharma.u-nancy.fr).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 11 April 2001; accepted in final form 5 June 2001.
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Am J Physiol Heart Circ Physiol 281(4):H1476-H1480
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ABSTRACT
INTRODUCTION
