www.elsevier.com/locate/brainres

Brain Research 1021

Research report

Alteration in dendritic morphology of pyramidal neurons from the

prefrontal cortex of rats with renovascular hypertension

Elenia Vegaa, Maria de Jesus Gomez-Villalobosb, Gonzalo Floresb,*

aEscuela de Biologıa. Universidad Autonoma de Puebla, Puebla, MexicobLaboratorio de Neuropsiquiatrıa, Instituto de Fisiologıa. Universidad Autonoma de Puebla, 14 Sur 6301, San Manuel, Puebla 72570, Mexico

Accepted 27 June 2004

Abstract

We have studied, in the rat, the dendritic morphological changes of the pyramidal neurons of the medial part of the prefrontal cortex

induced by the chronic effect of high blood pressure. Renovascular hypertension was induced using a silver clip on the renal artery by

surgery. The morphology of the pyramidal neurons from the medial part of the prefrontal cortex was investigated in these animals. The blood

pressure was measured to confirm the increase in the arterial blood pressure. After 16 weeks of increase in the arterial blood pressure, the

animals were sacrificed by overdoses of sodium pentobarbital and perfused intracardially with a 0.9% saline solution. The brains were

removed, processed by the Golgi–Cox stain method and analyzed by the Sholl method. The dendritic morphology clearly showed that the

hypertensive animals had an increase (32%) in the dendritic length of the pyramidal cells with a decrease (50%) in the density of dendritic

spines when compared with sham animals. The branch-order analysis showed that the animals with hypertension exhibit more dendritic

arborization at the level of the first to fourth branch order. This result suggests that renovascular hypertension may in part affect the dendritic

morphology in this limbic structure, which may implicate cognitive impairment in hypertensive patients.

D 2004 Elsevier B.V. All rights reserved.

Theme: Disorders of the nervous system

Topic: Neuropsychiatric disorders

Keywords: Renovascular hypertension; Dendrite; Golgi–Cox stain; Medial part of the prefrontal cortex; Pyramidal neuron

1. Introduction

Hypertension is an important risk factor for cerebro-

vascular disease causing brain damage with the develop-

ment of vascular cognitive impairment and vascular

dementia [2,3,5,15,28,41,44,45,51]. Disruption of the

blood–brain barrier is thought to contribute to these

disorders [1,10]. Several studies in animal models of

hypertension have demonstrated that chronic elevated

blood pressure may produce brain changes such as brain

atrophy, loss of nerve cells in cerebrocortical areas, and

0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.brainres.2004.06.042

* Corresponding author. Tel.: +522 244 1657; fax: +522 233 4511.

E-mail address: gflores@siu.buap.mx (G. Flores).

glial reaction [2,3,5,15,28,35,41,44,51]. In-vivo imaging

studies in patients with essential hypertension have

corroborated these brain changes [28,56]. In addition,

antihypertensive treatment with Ca2+ antagonists showed a

protective effect on brain damage caused by hypertension

[2,15]. However, the progressive decline in the cognitive

function associated with the hypertension is not well

understood. Several studies suggest that the prefrontal

cortex is involved with the cognitive processes, particularly

learning and memory [9,16,22,56]. Lesions of the supra-

limbic area of the medial part of the prefrontal cortex may

alter the memory and learning [16,22]. The medial part of

the prefrontal cortex is interconnected via glutamatergic

projections [24,49] with the ventral hippocampus and with

various other limbic cortexes via intracortical projections

(2004) 112–118

E. Vega et al. / Brain Research 1021 (2004) 112–118 113

[21,24]. Hippocampal neuronal activity exerts an important

regulatory control on the medial part of the prefrontal

cortex cells [14,18,34]. Synapses from the hippocampus to

the medial part of the prefrontal cortex are modifiable

synapses and may express different forms of plasticity in

all cognitive processes [10,22,52]. Recent studies related

the synaptic plasticity in the hippocampal–prefrontal cortex

pathway with two specific aspects of learning and memory,

i.e. the consolidation of information and working memory

[7,22,31].

Morphological studies of the pyramidal neurons of

medial part of the prefrontal cortex using a hypertensive

animal model, caused by using a clip in the thoracic aorta,

may in part help to understand the cognitive changes

resulting from chronic high blood pressure. Our inves-

tigation was designed to assess whether chronic hyper-

tension affects the dendritic length and spine density on

pyramidal neurons of layer 3 of the medial part of the

prefrontal cortex. The dendritic morphology clearly

showed that animals with renovascular hypertension had

an increase in the dendritic length of the pyramidal

neurons with a decrease in the density of dendritic spines

when compared to sham animals. The results suggest an

interesting and important effect of chronic hypertension on

these pyramidal cells.

Fig. 1. Graph of the systolic pressure of the renovascular hypertension

animal. The systolic pressure was increased in the animals with hyper-

tension. Closed circles indicate the meanFS.E.M. (n=10) from hyper-

tensive animals and the open circles correspond to the mean and FS.E.M.

(n=10) from sham rats. (*P b0.01).

2. Material and methods

2.1. Animals

Male Wistar rats (300–350 g) were obtained from our

animal facility. Animals were individually housed in a

temperature- and humidity-controlled environment in a 12–

12 h light–dark cycle with free access to food and water.

Renovascular hypertension was induced by a 0.2-mm

internal diameter, silver clip. Under chloral hydrate

anesthesia (360 mg/kg, i.p.), the left renal artery was

occluded by the silver clip. Sham rats underwent a similar

procedure with manipulation of the left renal artery but

without permanent attachment of the clip. All surgical

procedures described in this study are in accordance with

the bGuide for the Care and Use of Laboratory AnimalsQof the Mexican Council for Animal Care as approved by

the BUAP Animal Care Committee. All efforts were made

to minimize animal suffering and to reduce the number of

animals used.

2.2. Measure of the blood pressure

One week before and after use of the clip on the left

renal artery, the blood pressure was measured in the

renovascular hypertensive (n=10) and sham (n=10)

animals. From the second week, the blood pressure

was measured every 2 weeks for 16 weeks. Systolic

and diastolic blood pressures were measured by the tail-

cuff method (XBP1001 Rat tail, Blood Pressure system,

Kent Scientific). Systolic blood pressures (meanFS.E.,

mm Hg) for the sham rats and age-matched renovas-

cular hypertensive rats were measured as previously

described [39].

2.3. Golgi–Cox stain

Immediately after the last blood pressure measurement

(16 weeks after the clip attachment), the rats (n=10 per

group) were deeply anesthetized with sodium pentobarbital

and perfused intracardially with 0.9% saline solution. The

brains were removed and processed by Golgi–Cox stain-

ing, by using procedures described previously [42,43,52].

The brains were first stored in the dark for 14 days in

Golgi–Cox solution then 3 days in 30% sucrose. The

brains were cut into 200-Am-thick sections on the coronal

plane at the level of the medial part of the prefrontal cortex

[37] by using a vibratome. Sections were collected on

cleaned, gelatin-coated microscope slides (four sections/

slide) and stained with ammonium hydroxide for 30 min,

followed by Kodak Film Fix for another 30 min, and then

washed with water, dehydrated, cleared, and mounted

using a resinous medium.

The Golgi-impregnated pyramidal neurons of the

medial part of the prefrontal cortex were readily identified

by their characteristic triangular soma shape, apical

dendrites extending toward the pial surface, and numerous

dendritic spines. The criteria used to select neurons for

reconstruction were essentially as was described previ-

ously [43,52]; location of the cell soma in layer 3 of the

medial prefrontal cortex; full impregnation of the neurons,

presence of at least three primary, basilar dendritic shafts,

each of which branched at least once, and no morpho-

logical changes attributable to Golgi–Cox stain. Five

neurons in each hemisphere (10 neurons per animal) were

drawn using camera lucida at a magnification of 250�(DMLS, Leica Microscope) by a person who was not

Fig. 2. Photomicrograph illustrating Golgi–Cox-impregnated dendrite

arborization and dendrite spines on medial–prefrontal–cortex–layer 3

pyramidal neurons of sham rats (A,B) and animals with renovascular

hypertension (C,D).

Fig. 4. Total dendritic length from medial–prefrontal–cortex–layer 3

pyramidal neurons of renovascular hypertensive animals (n=100 neurons

per group). The dendritic length was increased in the hypertensive animals

(*P b0.01).

E. Vega et al. / Brain Research 1021 (2004) 112–118114

knowledgeable of the surgery conditions. For each neuron,

the dendritic tree, including all branches, was recon-

structed and the dendritic tracing was quantified by Sholl

analysis [50]. The dendritic surface was quantified by

counting the number of branches at each order from the

cell body by Sholl analysis [43,52], and by counting the

number of ring intersections using an overlay of concen-

tric rings (10 Am between rings). The density of dendritic

spines was measured from the basal dendrites by drawing

at least 10-Am-long segments from close to the cell body

and from the terminal tips at high power (1000�) and

counting the number of spines.

Fig. 3. Spine density of medial–prefrontal–cortex–layer 3 pyramidal

neurons of renovascular hypertensive (n=100 neurons) or sham animals

(n=100 neurons). Both proximal and distal spine density were decreased in

the hypertensive animals when compared with the sham controls

(*P b0.01).

Data from the Sholl analyses and the spine densities were

analyzed using a two-tailed Kruskal–Wallis and Mann–

Whitney tests (Pb0.05 was considered significant).

3. Results

3.1. Blood pressure

Control blood pressure was measured before the attach-

ment of the clip by surgery and no differences in the systolic

blood pressure were measured between the sham and

hypertensive rats (Fig. 1). Two weeks after the application

of the clip, there was an increase the systolic blood pressure

in the renovascular hypertensive rats when compared with

the sham animals (Pb0.01) (Fig. 1).

3.2. Dendritic morphology

Dendritic branching and density of dendritic spines of

neurons (100 neurons per group) of the medial part of the

prefrontal cortex were measured by Golgi–Cox stain for

both hypertensive and sham rats. Maximum branch order,

spine density, and total dendritic length obtained were

similar to our previous report [52]. The Golgi–Cox

impregnation procedure clearly filled the dendritic shafts

Fig. 5. Sholl analysis of dendrites of medial–prefrontal–cortex–layer 3

pyramidal neurons. Closed circles indicate the meanFS.E.M. (n=100

neurons) of hypertensive animals and the open circles correspond to the

meanFS.E.M. (n=100 neurons) of sham rats. The group of animals that

developed hypertension showed an increase in the dendritic length when

compared with the sham-control (*P b0.01).

Fig. 6. Graphs of branch order of pyramidal neurons of layer 3 of the medial

prefrontal cortex from renovascular hypertensive animals. Closed circles

indicate the meanFS.E.M. (n=100 neurons from 10 rats) from animals with

hypertension and the open circles correspond to the meanFS.E.M. (n=100

neurons from 10 rats) from sham rats. The group of animals that developed

hypertension showed an increase in the dendritic arborization at the level of

the first to fourth branch order compared to the sham rats (*P b0.01 to first

to third order; **P b0.05 to fourth order).

E. Vega et al. / Brain Research 1021 (2004) 112–118 115

and spines of layer 3 of the pyramidal medial part of the

prefrontal cortex neurons (Fig. 2). Comparisons between

hypertensive and sham animals showed that the mean spine

density of the dendrites of pyramidal neurons of layer 3 of

the medial part of the prefrontal cortex in the hypertensive

animals were lower than their controls (48% and 51%

decrease in the proximal and distal dendritic spines from the

body of the neuron, Pb0.001) (Figs. 2 and 3).

As measured by Sholl analysis, total dendritic length of

the medial part of the prefrontal cortex neurons differed

significantly (Pb0.001) between hypertensive and sham

rats (Fig. 4). Interestingly, the hypertensive animals showed

an increased in the dendritic length (Pb0.001). The analysis

of intersection per radius of shell shows that the hyper-

tensive animals had more intersections per shell or more

dendritic arborization than the sham rats (P=0.03) (Fig. 5).

In addition, the branch-order analysis also suggests that the

hypertensive rats had more dendritic arborization with an

increase in the first to the fourth branch order in comparison

with the sham animals (Pb0.01 to the first to third order;

Pb0.05 to the fourth order) (Pb0.01) (Fig. 6).

4. Discussion

Our aim was to investigate the consequences of 16

weeks of high blood pressure, induced by the occlusion of

a renal artery by a silver clip, on the basilar, dendritic-

structural morphology of layer 3 pyramidal cells of the

prefrontal cortex. We found that hypertension causes major

reductions in dendritic spine density with increases in the

dendritic length in layer 3 pyramidal neurons of the medial

part of the prefrontal cortex and these data may be linked

in part with the cognitive impairment seen in hypertension.

The high levels of systolic pressure caused by the

occlusion of a renal artery, reported here, are consistent

with previous reports [2,25,39,55] using the same proce-

dure. In those studies, hypertensive rats showed a clear

increase in the systolic blood pressure with an alteration in

cognition [41].

Several reports support a causal role of hypertension in the

cognitive decline in hypertensive patients [2,3,28,41]. Hyper-

tension produces changes in the brain, such as vascular

remodeling, impaired cerebral autoregulation, white-matter

lesions, and cerebral microbleed [2,3,28]. Furthermore,

hypertension has been implicated in vascular dementia

[2,41]. Evidence has demonstrated that the cognitive func-

tions are regulated by the prefrontal cortex [7,8,9,16,56].

Participation of the medial prefrontal cortex in cognition is

well recognized when its connections are analyzed. The

medial prefrontal cortex receives and sends excitatory

projections to the CA1 region from the hippocampus

[21,24,49], a critical structure in memory [7,16]. The layer

3 of the medial prefrontal cortex may be regulated by

hippocampal projections [12,21,24,48,49] and its activity

may be modulated by synaptic inputs from the hippocampus

[17,18,33,34]. The nucleus accumbens sends signals to the

ventral pallidum [58], a critical structure in emotions [42,43],

and the ventral pallidum sends signals to the thalamus [58].

Our results clearly show that renovascular hypertension

produces alteration in the morphology of the dendrites of the

pyramidal neurons. Exactly how renovascular hypertension

came to enhance the dendritic arborization at the level of the

first to fourth branch orders of the pyramidal neurons of the

medial part of the prefrontal cortex is not clear. However, as

mentioned, renovascular hypertension is associated with

vascular remodeling, and physiological studies have shown

that renovascular hypertension is associated with a dysfunc-

tional endothelium caused by deficient production of nitric

oxide (NO) derived from the endothelium [29], which alters

the vasodilatation in this model [25,55]. At the level of the

neurons, the activity of the enzyme that catalyzes the

production of NO, nitric oxide synthase, is decreased in

animals with renovascular hypertension when compared to

sham animals [20]. Interestingly, a recent report analyzed

the activity of the nitric oxide synthase in different regions

of the brain, before and after establishing hypertension in

rats [40], and the authors suggest that when hypertension

exists the activity of the nitric oxide synthase is enhanced in

the hypothalamus and brainstem. The hypothalamus is a

critical structure in the control of hormones, e.g. the

production of corticosterone is controlled by an adrenocor-

ticotropic-hormone, which is regulated by a corticotropine-

releasing hormone [26]. The corticotropine-releasing hor-

mone is produced by the hypothalamus. The corticosterone

per se also may affect the density of dendritic spines [26].

High levels of corticosterone may produce a decrease in the

density of dendritic spines in pyramidal neurons of the

hippocampus [26]. In addition, several recent studies have

shown that the nitric oxide per se may in part be regulating

the dendritic spines and branching in the pyramidal cells of

the cortex [4,30,32,38,46,47]. An increase in the activity of

the NO may explain the increase in the dendritic length

[4,30,38].

E. Vega et al. / Brain Research 1021 (2004) 112–118116

The pyramidal neurons of the medial prefrontal cortex

used glutamate as the neurotransmitter. Spine creation and

destruction at glutamatergic synapses is largely controlled

by glutamate itself [36]. Some types of glutamate receptors,

such as N-methyl-d-aspartic acid (NMDA) or metaboli-

tropic receptors, activate phosphorylation of skeletal micro-

tubular protein and influence synaptic maturation, spine

morphology, and possibly the growth of new spines

[19,36,54]. Perhaps dendritic development is dependent on

NMDA receptor activity, and then the asymmetric synapse

density in striatal neurons (formed by glutamatergic input)

dramatically declines by NMDA blockade in neonatal rats

[22,23,54], whereas the dendritic spines of the cortical

neurons are sites of the majority of excitatory synapses and

are associated with long-term synaptic plasticity and are

inhibited by the activation of the NMDA receptors [13].

Glutamate is also a potent neurotoxin and may, under certain

circumstances, produce neural damage and possible spine

elimination [53,54]. Some studies have demonstrated that

the NMDA receptors may mediate the spinal sympathetic

reflexes, which initiate episodic hypertension after a spinal

cord injury [27,59], consistent with this relation between the

glutamate transmission and hypertension. In addition, the

rostral ventrolateral medulla neurons of animals with

renovascular hypertension exhibited an increased response

to glutamate actions [6]. Rilmenidine, a second-generation,

centrally acting, antihypertensive drug, with a hypotensive

effect, is dependent on functional NMDA receptors [59]. All

this data, taken together with a recent report, showed the

activation of NMDA receptors and subsequent release of

nitric oxide may in part trigger the growth of presynaptic

phylopodia, which play an important role in synaptogenesis

and spine formation [32]. Pisu et al. [38] have demonstrated

the NO-glutamate interactions via NMDA receptors in the

development of the dendritic tree of the Purkinje neurons.

One can hypothesize that the alteration of the activity of the

nitric oxide, together with altered response to glutamate,

especially via an NMDA receptor, may in part participate in

the morphological changes found in the dendritic pyramidal

neurons of the medial part of the prefrontal cortex from the

rats with renovascular hypertension. There is a need for

further studies, to relate the NO and glutamate activity in the

medial part of the prefrontal cortex at different times after

establishment of hypertension in rats, to clarify all these

explanations.

Another possibility is that the loss of the inputs to the

medial part of the prefrontal cortex may produce a decrease

in the spine dendrites in the pyramidal neurons by loss of

the synapse on spines [11,23]. However, under some

physiological conditions this is not true, e.g. during the

estrous cycle there is a decrease of the spine density of the

pyramidal neurons of the area CA1 of the hippocampus [57]

without loss of the inputs. Another possibility is that chronic

hypertension may alter the integrity of the blood–CSF

barrier in addition to the vascular alteration in the brain [1],

and several toxins may cross this barrier with a toxic effect

on the dendritic morphology [11]. It is tempting to speculate

that the damage of the blood–CSF barrier may result in a

decrease in the dendritic spine density. The increase in the

total dendritic length with the decrease in the dendritic spine

is especially intriguing, because this may be caused by an

ineffective mechanism of the synaptic connectivity in the

medial prefrontal cortex.

In summary, our findings provide evidence for a decrease

in dendritic spines with an increase in the arborization of the

pyramidal neurons of the medial part of the prefrontal cortex

as a result of chronic renovascular hypertension. Given the

functional role and the interconnection of the medial part of

the prefrontal cortex with other cognitive structures such as

hippocampus and nucleus accumbens, these morphological

changes reported here may contribute to the explanation of

some cognitive data reported in hypertensive patients.

Acknowledgements

This study was supported in part by grants from

CONACyT-Mexico (No. 40664) and VIEP-BUAP (No. IV-

34-04/SAL/G to GF). We are grateful to Dr. Carlos Escamilla

for his help and suggestions related to keeping of animals.

EV is a student of BUAP. MJG and GF are members of the

Researcher National System of Mexico. Thanks to Dr. Ellis

Glazier for editing the English-language text.

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