Middle cerebral artery alterations in a rat chronic hypoperfusion model






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Middle cerebral artery alterations in a rat chronic hypoperfusion model

Ana Márquez-Martín,1Francesc Jiménez-Altayó,1Ana P. Dantas,2Laura Caracuel,1Anna M. Planas,3 and Elisabet Vila1

1Departament de Farmacologia, Terapèutica i Toxicologia, Institut de Neurociències, Facultat de Medicina, Universitat Autònoma de Barcelona, Bellaterra; and2Institut Clínic del Tòrax and3Departament d’Isquèmia Cerebral i

Neurodegeneració, Institut d’Investigacions Biomèdiques August Pi i Sunyer, Barcelona, Spain Submitted 5 August 2011; accepted in final form 16 November 2011

Márquez-Martín A, Jiménez-Altayó F, Dantas AP, Caracuel L, Planas AM, Vila E.Middle cerebral artery alterations in a rat chronic hypoperfusion model.J Appl Physiol112: 511–518, 2012. First published November 17, 2011; doi:10.1152/japplphysiol.00998.2011.—Chronic cere-bral hypoperfusion (CHP) induces microvascular changes that could contribute to the progression of vascular cognitive impairment and dementia in the aging brain. This study aimed to analyze the effects of CHP on structural, mechanical, and myogenic properties of the middle cerebral artery (MCA) after bilateral common carotid artery occlusion (BCCAO) in adult male Wistar rats. Sham animals underwent a similar surgical procedure without carotid artery (CA) ligation. After 15 days of occlusion, MCA and CA were dissected and MCA structural, mechanical, and myogenic properties were assessed by pressure myography. Collagen I/III expression was determined by immunofluorescence in MCA and CA and by Western blot in CA. mRNA levels for 1A1, 1A2, and 3A1 collagen subunits were quan-tified by quantitative real-time PCR in CA. Matrix metalloproteinase (MMP-1, MMP-2, MMP-9, and MMP-13) and hypoxia-inducible factor-1␣ (HIF-1␣) protein expression were determined in CA by Western blot. BCCAO diminished cross-sectional area, wall thick-ness, and wall-to-lumen ratio. Nevertheless, whereas wall stress was increased, stiffness was not modified and myogenic response was diminished. Hypoperfusion triggered HIF-1␣ expression. Collagen I/III protein expression diminished in MCA and CA after BCCAO, despite increased mRNA levels for 1A1 and 3A1 collagen subunits. Therefore, the reduced collagen expression might be due to proteo-lytic degradation, since the expression of MMP-1 and MMP-9 in-creased in the CA. These data suggest that BCCAO induces hypo-trophic remodeling by a mechanism that involves a reduction of collagen I/III in association with increased MMP-1 and MMP-9 and that decreases myogenic tone in major arteries supplying the brain. bilateral common carotid artery occlusion; cerebral blood flow; hyp-oxia; vascular remodeling; myogenic response

BLOOD FLOW IRRIGATIONto the brain is tightly regulated to ensure

correct neuronal function. Nevertheless, several stroke risks factors can alter this homeostasis and lower cerebral blood flow (CBF) below a cerebral hypoperfusion threshold where cell function is compromised (14). Long-lasting reduction of CBF, known as chronic cerebral hypoperfusion, elicits multiple al-terations, such as induction of oxidative stress (1, 37) and neuronal death (37), among others. In addition, experimental evidence suggests that cerebral hypoperfusion is associated with the decline of cognitive function and the development of several types of dementia (12, 16).

The middle cerebral artery (MCA) is one of the most extensive brain territories of irrigation (18), and reductions in

MCA flow have been associated with cognitive disorders (19, 47). To better understand the effects of blood flow reduction in cerebrovascular disorders, a rat model of cerebral hypoperfu-sion has been well established by bilateral common carotid artery occlusion (BCCAO). Permanent BCCAO mimics some of the clinical conditions of cerebrovascular hypoperfusion associated with aging in humans (13, 16) and contributes to behavioral and cognitive deficits (48, 49, 55). In this animal model, the cerebrovascular flow drops sharply immediately after occlusion (7) and persists for several days after permanent BCCAO (36). However, compensatory redistribution via the vertebrobasilar network progressively recovers blood flow in almost all brain regions, and CBF values are indistinguishable from controls after 1 mo (7, 36).

Vascular remodeling involves structural changes in the ves-sel wall in response to hemodynamic stimuli. These alterations in composition/size of the vessel allow adaptation and repair (41). In resistance arteries, chronic increase or decrease in blood flow induces outward or inward remodeling, respectively (5, 15, 27, 40). The mechanisms involved in flow-induced remodeling have been mainly investigated in large and small arteries outside the brain. Nevertheless, previous studies have documented an enlargement and tortuosity of the basilar artery in response to permanent BCCAO (25, 44, 54). In addition, after 6 mo of BCCAO, the majority of main intracerebral arteries, including the MCA, increase their diameter, possibly contributing to the normalization of CBF (7). However, vas-cular remodeling is a complex process that usually takes place within hours to several days of the initiating stimulus (31). Thus a decrease of blood flow has been described to induce vascular remodeling in a period as short as 2 days (5). More-over, changes in myogenic capacity have also been reported to modify vessel properties. For example, a decrease in MCA myogenic response is a hallmark of cerebral ischemia after transient MCA occlusion (9, 22, 23). In the present study, we aimed to analyze the effect of 15 days of BCCAO on structural, mechanical, and myogenic cerebrovascular properties and to determine potential mechanisms involved in those alterations.


Animals.Thirteen- to 15-wk-old male Wistar rats (Charles River, Barcelona, Spain) were housed in a temperature-controlled room on a 12:12-h light-dark cycle and provided with access to food and water ad libitum. The investigation conforms to the “Guide for the Care and Use of Laboratory Animals” published by the U.S. National Institutes of Health (NIH Publication No. 85-23, revised 1996). Approval for this work was granted by the Universitat Autònoma de Barcelona Ethical Committee.

Bilateral common carotid artery occlusion.Rats were anesthetized by inhalation with 5% isoflurane in 0.5–1 l/min O2 and maintained

with 2–2.5% isoflurane in 0.5–1 l/min O2. Adequacy of anesthesia Address for reprint requests and other correspondence: E. Vila, Departament

de Farmacologia, Terapèutica i Toxicologia, Facultat de Medicina, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain (e-mail: elisabet.vila @uab.es).


was confirmed by total absence of reflex responses (pinching the tail, pedal withdrawal, and pupilar reflex). Body temperature was moni-tored using a rectal probe and maintained at 37 ⫾ 0.5°C during surgery. To produce analgesia, meloxicam (2 mg/kg) was adminis-tered subcutaneously 30 min before anesthesia and 24 and 48 h after surgery (1 mg/kg). Once the ventral neck was shaved and washed with 10% povidone iodine solution, BCCAO was performed as previously described (25, 52). Briefly, a ventral midline incision was made in the neck to expose both common carotid arteries (CA). Both CA were carefully separated from the vagus nerve, and occlusions were per-formed with a 5-0 silk suture (Suturas Aragó, Barcelona, Spain). The wound was closed, and the rats were allowed to recover from anesthesia and placed back into their cages. Sham animals underwent a similar surgical procedure without CA ligation. After 15 days, animals were killed by an overdose of sodium pentobarbitone for tissue harvesting.

Tissue preparation.The entire brain and segments of CA upstream from the silk suture were removed and placed in ice-cold physiolog-ical salt solution (PSS) of the following composition (in mM): 112.0 NaCl, 4.7 KCl, 2.5 CaCl2, 1.1 KH2PO4, 1.2 MgSO4, 25.0 NaHCO3,

and 11.1 glucose, maintained at 4°C and gassed with 95% O2and 5%

CO2. For pressure myography studies, the MCA was dissected out of

the brain under a surgical microscope, cleaned from surrounding tissue, and kept in cold PSS. For immunofluorescence studies, CA and MCA were fixed with 4% phosphate-buffered paraformaldehyde (PFA; pH 7.4) for 45 min and washed three times with phosphate-buffered saline (PBS; pH 7.4). After clearing, vessels were placed in PBS containing 30% sucrose overnight, transferred to a cryomold containing Tissue Tek optimal cutting temperature (OCT) embedding medium, and frozen in liquid nitrogen. For Western blot and RT-PCR studies, the CAs were quickly frozen in liquid nitrogen. All samples frozen in liquid nitrogen were kept at ⫺70°C until the day of the experiment.

Pressure myography.Structural, mechanical, and myogenic prop-erties of the MCA were studied with a pressure myograph (Danish Myo Tech, model P100; J.P. Trading, Aarhus, Denmark), as previ-ously described (22, 23). Briefly, vessels were placed on two glass microcannulas and carefully adjusted so that the vessel walls were parallel without stretching. Intraluminal pressure was then raised to 140 mmHg, and the artery was unbuckled by adjusting the cannulas. Afterwards, the artery was left to equilibrate for 1 h at 70 mmHg in gassed PSS (37°C). Intraluminal pressure was reduced to 10 mmHg, and a pressure-diameter curve (10 –120 mmHg) was obtained. Internal and external diameters (DiCaandDeCa) were measured for 3 min at each intraluminal pressure. The MCA was then set to 70 mmHg and allowed to equilibrate for 30 min at 37°C in gassed calcium-free PSS (0 Ca2⫹: omitting calcium and adding 10 mM EGTA; Sigma-Aldrich,

St. Louis, MO), and a second pressure-diameter curve was obtained in 0 Ca2⫹-PSS (passive conditions). Finally, the artery was fixed (70

mmHg) with 4% PFA for 45 min and prepared for immunofluores-cence assays as indicated above.

Structural parameters such as wall thickness (WT), cross-sectional area (CSA), and wall-to-lumen ratio (W/L) were calculated as fol-lows: WT⫽(De0Ca⫺Di0Ca)/2, CSA⫽(␲/4)[(De0Ca)2⫺(Di0Ca)2],

and W/L⫽(De0Ca⫺Di0Ca)/2Di0Ca, whereDe0CaandDi0Caare the

external and internal diameter observed for a given intravascular pressure in passive conditions (0 Ca2⫹-PSS), respectively.

Mechani-cal parameters were Mechani-calculated as described by Baumbach and Heistad (1989). Circumferential wall strain (ε) was calculated as (Di0Ca ⫺ Do0Ca)/Do0Ca, where Do0Ca is the internal diameter at 10 mmHg,

measured under fully relaxed conditions (0 Ca2⫹-PSS).

Circumferen-tial wall stress (␴) was calculated as (P⫻Di0Ca)/2WT, were P is the

intraluminal pressure (1 mmHg ⫽ 133.4 N/m2) and WT is wall

thickness at each intraluminal pressure in 0 Ca2⫹medium. Elastic

modulus was calculated by fitting strain-stress data to␴ ⫽ ␴origexp

(␤ε), where␴origis the stress at the original diameter (10 mmHg). The

␤value was used as an index of wall stiffness (33).

Percentages of myogenic response were determined in each pres-sure using the following formula: 100⫻DiCa/Di0Ca, whereDiCaand

Di0Caare the internal diameters measured in active (2.5 mM Ca2⫹

-PSS) and passive (0 Ca2⫹-PSS) conditions, respectively. Slopes of the

myogenic response-pressure curves for individual vessels were deter-mined by linear regression.

Immunofluorescence.Frozen transverse sections (14␮m) of MCA or CA were placed on gelatin-coated slides and air-dried (90 min). Sections were incubated with a rabbit primary polyclonal antibody against collagen I/III (1:30; Calbiochem, San Diego, CA). After being washed, rings were incubated with the secondary donkey anti-rabbit IgG antibody conjugated to Cy3 (1:200; Jackson ImmunoResearch Laboratories, West Grove, PA). Immunofluorescent signals were viewed by fluorescent laser confocal microscopy (Leica TCS SP2) using a ⫻20 (CA) or⫻63 (MCA) objective. The Cy3-labeled anti-body was visualized by excitation at 561 nm and detection at 600 –700 nm. The specificity of the immunostaining was evaluated by omission of the primary antibody and processed as described above. Under these conditions, no staining was observed in the vessel wall in any experimental situation. Quantitative analysis of collagen I/III fluores-cence was performed with MetaMorph 4.6 image analysis software (Universal Imaging, Molecular Devices, Downingtown, PA). The intensity of fluorescence per area was calculated in three rings of each animal, and the results were expressed in arbitrary units. All measure-ments were conducted blindly.

Carotid artery morphometry.Morphometric determination of the lumen, media, and vessel areas of the CA was performed using MetaMorph 4.6 image analysis software, as previously described (50). To determine the luminal area, the CSA enclosed by the internal elastic lamina was corrected to a circle by applying the form factor

l2/4to the measurement of the internal elastic lamina, wherelis the

length of the lamina. The vessel area was determined by the CSA enclosed by the external collagen, corrected to a circle by applying the same form factor (l2/4) to the measurement of the external collagen.

The CSA was calculated as follows: CSA⫽leli, whereleandliare the vessel and luminal area, respectively. Mean values were obtained from at least two different sections from the same artery.

Western blot analysis. Equal amounts of proteins (50 ␮g) from homogenized CA were loaded into individual lanes of minigels of 4 –12% polyacrylamide gels (Novex precast TBE gels; Invitrogen, Barcelona, Spain), transferred onto nitrocellulose membrane, and blocked in PBS-Tween 20 (0.1% vol/vol) containing 5% nonfat milk overnight at 4°C. Western immunoblotting was performed with monoclonal antibodies against MMP-1 (1:1,000; Calbiochem) and MMP-9 (1:1,000; Abcam, Cambridge, UK) or with polyclonal anti-bodies against collagen I/III (1:500; Calbiochem), MMP-13 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA), MMP-2 (1:1,000; Ab-cam), and HIF-1␣(1:1,000; Novus Biologicals, Littleton, CO). After being washed, membranes were incubated with peroxidase-conju-gated secondary antibodies goat anti-mouse (1:2,000, for MMP-1 and MMP-9; Thermo Fisher Scientific, Barcelona, Spain), goat anti-rabbit (1:2,000, for collagen I/III, MMP-2, and HIF-1␣; Thermo Fisher Scientific), and donkey anti-goat (1:2,000, for MMP-13; Santa Cruz Biotechnology). Immunocomplexes were detected using an enhanced horseradish peroxidase chemiluminescence system (SuperSignal West Pico chemiluminescence substrate; Thermo Fisher Scientific). The signal intensity of the bands was quantified using ImageJ software (National Institutes of Health, Bethesda, MD). All membranes were reblotted using monoclonal antibody anti-GAPDH (1:2,500; Santa Cruz Biotechnology) as a loading control. Data were normalized to corresponding values of GAPDH densitometry.

Real-time quantitative RT-PCR. Total RNA (tRNA) was isolated using Trizol (TRI Reagent) from frozen CA according to the manu-facturer’s protocol. tRNA quality was evaluated by electrophoresis on a 1% denaturing agarose gel; tRNA concentration and purity were determined by measuring absorbance at 260 and 280 nm (A260/A280) by spectrophotometry (NanoDrop ND-1000; Thermo Fisher


Scien-tific). The samples with intact ribosomal RNA bands and with an A260/A280 ratio between 1.8 and 2.0 were used for cDNA synthesis. tRNA (1␮g) was inversely transcribed to cDNA using a commercial kit (High-Capacity cDNA reverse transcription kit; Applied Biosys-tems, Foster City, CA) following the manufacturer’s conditions in 20

␮l of volume reaction containing reaction buffer, deoxynucleoside triphosphate (dNTPs), random primers, reverse transcriptase (Multi-Scribe reverse transcriptase), and DEPC-treated water. The reaction took place in the thermocycler (Peltier Thermal Cycler PTC-200) for 10 min at 25°C (annealing step), followed by an extension step at 37°C for 2 h and a final step at 85°C for 5 min for heat inactivation. Gene expression for collagen subunits 1A1, 1A2, and 3A1 were quantified by quantitative real-time PCR (qRT-PCR) based on SYBR green fluores-cence using the 18S ribosomal subunit of RNA (Hs99999901 s1) as internal control with a GeneAmp 7500 PCR system (Applied Biosys-tems) following the manufacturer’s conditions: first step of polymerase activation at 95°C for 10 min and 40 cycles composed of two steps, denaturing at 95°C for 15 s and annealing/extension at 60°C for 1 min. The specific primer sequences for rodent were 1A1 (forward: 5= -AGAGTACTGGATCGACCCTAACCA-3= and reverse: 5= -CAG-GTCTGACCTGTCTCCATGTT-3=), 1A2 (forward: 5= -CTACTGGT-GAAACCTGCATCCA-3= and reverse: 5= -GGGCGCGGCTGTAT-GAG-3=), and 3A1 (forward: 5=-GGATCTGTCCTTTGCGATGAC-3= and reverse: 5=-GCTGTGGGCATATTGCACAA-3=). Primers were de-signed from conserved regions of the studied genes: 1A1 (NM_007742.3), 1A2 (NM_007743.2), and 3A1 (NM_009930.2). qRT-PCR reactions were set following the manufacturer’s conditions. Thresh-old cycle (Ct) values obtained for each gene were referenced to r18S (⌬Ct) and converted to the linear form using the term 2⫺⌬Ctas a value

directly proportional to the copy number of cDNA and initial quantity of mRNA (35).

Statistical analysis.Results are means⫾SE of the number (n) of rats indicated. The dependence of structural, mechanical, or myogenic properties on BCCAO was assessed by two-way analysis of variance (ANOVA) with repeated measures on the pressure factor. In the case of one single factor, unpaired Student’st-test was used. Data analysis was carried out using GraphPad Prism 4 software. A value of P

0.05 was considered significant.


Body weight.As shown in Table 1, body weight atday 0was similar in both groups of rats. BCCAO induced a decrease (P⬍0.05) of body weight on the first 2 days following surgery that recovered to the levels of sham animals by day 5. After-wards, a similar progression in body weight increase was observed in both groups of rats.

HIF-1.BCCAO caused sustained hypoxia in the vessels as assessed by the increased protein expression of HIF-1␣in CA from BCCAO animals at day 15 (Fig. 1). HIF-1␣ is a tran-scription factor controlling signaling cascades that are involved in vascular remodeling (26).

Structural and mechanical properties of the MCA. Diame-ters of the MCA were measured over the pressure range (10 –120 mmHg) under fully relaxed conditions (0 Ca2⫹-PSS),

and the influence of BCCAO on MCA structural properties is shown in Fig. 2. After 15 days of BCCAO, vessel and lumen diameters of the MCA were not significantly modified (results not shown). However, CSA (Fig. 2A), WT (Fig. 2B), and W/L (Fig. 2C) were decreased (P ⬍ 0.001) in BCCAO compared with sham rats. Analysis of the passive mechanical properties showed that wall stress (Fig. 3A) was increased (P⬍0.05) in MCA from BCCAO animals. Nevertheless, the stress-strain relationship (Fig. 3B) and the␤values were similar (sham: 9.6⫾ 1.2,n⫽10; BCCAO: 9.4 ⫾0.9,n⫽9), demonstrating that wall stiffness remained unchanged.

Myogenic properties of the MCA.The extent of the constric-tor tone, also called myogenic response, is shown in Fig. 3C. In BCCAO vessels, myogenic response was decreased at all of the perfusion pressures tested, indicating a loss of constrictor tone. Conversely, analysis of slopes of the myogenic response-pressure curves showed that this parameter was not altered in BCCAO (0.06 ⫾ 0.02, n⫽ 7) compared with sham vessels (0.08 ⫾0.03, n⫽ 10), indicating that myogenic reactivity is preserved after BCCAO.

Carotid artery morphometry.The morphometric analysis of rat CA demonstrated that CSA was decreased (P ⬍ 0.01) in BCCAO (1,716,612 ⫾ 272,610 ␮m2,n 5) compared with

sham vessels (3,245,512⫾ 318,869␮m2,n 4).

Expression of collagen I/III. Collagen I/III expression, shown as red fluorescence along the vessel wall of the MCA Table 1. Body weight progression in sham and BCCAO rats after surgery


Body Weight, g

Day 0 Day 1 Day 2 Day 5 Day 10 Day 15

Sham 305⫾5 297⫾5 302⫾8 306⫾5 329⫾4 344⫾4

BCCAO 314⫾5 281⫾5* 279⫾5* 301⫾5 319⫾6 340⫾5

Values are means⫾SE ofn⫽26 –30 sham or bilateral common carotid artery occlusion (BCCAO) rats. *P⬍0.05, sham vs. BCCAO by unpaired Student’s


Fig. 1. Representative immunoblot and densitometric analysis of hypoxia-inducible factor-1␣(HIF-1␣) protein expression in carotid arteries (CA) from sham and bilateral common carotid artery occlusion (BCCAO) rats. The expression of GAPDH as a loading control is also shown. Values are means⫾ SE ofn⫽9 rats. *P⬍0.05, sham vs. BCCAO by unpaired Student’st-test.


(Fig. 4A) and CA (Fig. 4B), was significantly reduced in both types of arteries from BCCAO rats compared with sham animals. The decrease of collagen I/III expression in MCA and CA after BCCAO was confirmed by Western blotting in CA (Fig. 5A). Analysis of mRNA levels for 1A1, 1A2, and 3A1 collagen subunits showed that all subunits were present in CA. Contrary to protein expression, BCCAO markedly enhanced mRNA expression of the 1A1 (Fig. 5B) and 3A1 (Fig. 5D) subunits (P⬍0.05), whereas results showed a decrease (P

0.05) in mRNA levels for the 1A2 subunit (Fig. 5C).

Protein expression of MMP.Expression of MMP-1 (Fig. 6A) and latent and active MMP-9 (Fig. 6B), analyzed by Western blotting, were increased (P ⬍ 0.05) in CA from BCCAO animals. In contrast, MMP-2 and MMP-13 expression re-mained unaltered (data not shown).


Permanent BCCAO in rats has been widely used as a model to study several cerebrovascular disorders (16, 51, 53). BC-CAO causes CBF decreases immediately after occlusion and is an interesting approach to evaluate the effects of cerebral hypoperfusion (7). Our findings show that permanent BCCAO induces an increase of HIF-1␣ expression, an endogenous marker of hypoxia that is sustained for at least 15 days, evidencing a chronic exposure to factors inducing vascular remodeling (26). BCCAO decreased the MCA wall material,

Fig. 3. Effect of BCCAO on mechanical and myogenic properties from rat MCA.A: wall stress-intraluminal pressure.B: stress-strain (Di/Do).C: myo-genic response. Do, internal diameter at 10 mmHg; Di, observed internal diameter from given intravascular pressure;DiCa, internal diameters measured in active (2.5 mM Ca2⫹-PSS) and passive (0 Ca2⫹-PSS) conditions,

respec-tively. Values are means⫾SE ofn⫽8 –10 rats. ***P⬍0.001; **P⬍0.01, sham vs. BCCAO by 2-way ANOVA.

Fig. 2. Effect of BCCAO on structural parameters from fully relaxed rat middle cerebral arteries (MCA).A: cross-sectional area (CSA)-intraluminal pressure.

B: wall thickness-intraluminal pressure.C: wall/lumen-intraluminal pressure. Values are means⫾SE ofn⫽8 –10 rats. ***P⬍0.001. sham vs. BCCAO by 2-way ANOVA.


and remodeling was paralleled by an increase in wall stress. Structural alterations of the MCA were accompanied by a decrease in collagen I/III protein expression by a mechanism that may involve increased degradation via MMP-1 and/or MMP-9. In addition, the myogenic response of the MCA was compromised. Therefore, we show alterations in the passive and active properties of an important cerebrovascular bed, which have not been, to our knowledge, previously evaluated after chronic cerebral hypoperfusion.

HIF-1 is a key transcriptor factor tightly regulated by cellu-lar oxygen concentration and is composed of␣- and␤-subunits (45). It regulates oxygen-dependent expression of target genes involved in proliferation, erythropoiesis, and angiogenesis,

among others (46). In the present study, we observed that after 15 days of permanent BCCAO there was an increase in CA HIF-1␣protein expression, corroborating tissue hypoxia in this vascular bed.

Mounting evidence has established that structural variations in cerebral vasculature have an important role in brain function (10), and therefore, a better understanding of the mechanisms involved in structural changes into those arteries are of partic-ular relevance and great clinical impact. In the present work, we focused on studying the effect of permanent BCCAO in MCA properties, because it is one of the longest intracranial arteries that irrigate an extensive territory in the brain, and decreases in MCA blood flow have been associated with

Fig. 4. Representative photomicrographs and quantification of immunofluorescence of confocal microscopic sections of MCA (A) and CA (B) from sham and BCCAO rats. ADV, adventitial layer; END, endothelial layer; IEL, internal elastic lamina; MED, media layer. Image size, 238⫻238␮m. Values are means⫾ SE ofn⫽7– 8 (MCA) orn⫽4 –5 (CA) rats. **P⬍0.01; ***P⬍0.001, sham vs. BCCAO by unpaired Student’st-test.

Fig. 5. Representative immunoblot and densitometric analysis of collagen (Col) I/III protein expression (A) and comparative analysis of mRNA levels of 1A1 (B), 1A2 (C), and 3A1 (D) subunits of collagen in CA from sham and BCCAO rats. Protein expression of GAPDH as a loading control is also shown. mRNA levels are expressed as 2⫺⌬Ctusing 18S as internal control. Values are meansSE ofn6 (protein expression) orn8 (mRNA levels) rats. *P0.05; ***P⬍0.001, sham vs. BCCAO by unpaired Student’st-test.


cognitive deficits (19, 47). It is feasible that alterations in MCA properties may be involved in the cognitive decline described in the BCCAO-model (48).

Our study shows a reduction in CSA and WT of the MCA after 15 days of permanent BCCAO. Although we did not find a significant alteration in MCA diameter after 15 days of permanent BCCAO, our study is consistent with the notion that permanent BCCAO induced physiologically important vascu-lar hypotrophic remodeling, since the decrease in the CSA was paralleled by a reduction in the W/L ratio of the MCA. The W/L ratio parameter allows for a better comparison of vessels, since it removes the effect of variability in vessel dimensions and is taken as an important predictor of cardiovascular events (32, 42). This observation is in agreement with previous studies in resistance arteries, where chronic decreases in blood flow induce hypotrophic remodeling (5, 40).

Wall stress has been postulated as an indicator of risk for brain injury (6, 8, 22). In the current study, the observed increase in wall stress correlates with a decrease in the CSA. It is well accepted that wall stress is the stimulus for subsequent growth (17), which is an adaptive response to preserve vessel integrity (3). We hypothesize that the increase of MCA wall stress observed after 15 days of permanent BCCAO occlusion might ultimately lead to MCA growth at later stages, likely mediated through the signaling cascades triggered by the ex-pression of HIF-1␣ observed here. Consistently, increase in MCA diameter has been observed after 6 mo of permanent BCCAO (7).

Different mechanisms have been involved in the reduction of wall material. Among others, changes in extracellular matrix (ECM) components are well documented (4). ECM is a dy-namic scaffold with the purpose of maintaining the integrity of blood vessels. We observed that after BCCAO, protein expres-sion of collagen type I and III, the most abundant in the vascular wall (34), decreased in the MCA. Furthermore, CA (i.e., the arteries occluded) also experienced a similar pattern of decrease in CSA and collagen I/III protein expression. The parallelisms of MCA and CA results summed to the very limited amount of sample we could obtain per vessel/animal drove us to perform the next sets of experiments for protein/ mRNA expression in CA only. Results from quantitative PCR

analysis for the expression of each subunit of collagen I and III in the vessel wall revealed an overall opposite effect compared with protein expression. Whereas both immunofluorescence and Western blot analysis showed a decrease of collagen I/III protein expression in animals with BCCAO, mRNA expression for collagen 1A1 and 3A1 were upregulated in those animals. Although mRNA expression of the 1A2 collagen subunit was downregulated following BCCAO, the low expression of this subtype (about 10,000 less than other subtypes) should not account for the decrease in collagen protein expression ob-served in this study. More likely, changes in posttranslational mechanisms or collagen degradation would contribute to these effects.

MMPs are endopeptidases important for the regulation of ECM dynamics and have been involved in ECM degradation. MMPs in the vessel wall are regulated by multiple stimuli, including hemodynamic forces such as low blood flow. We found that the decreases in collagen I/III deposition in MCA and CA were accompanied by an increase in MMP-1 and -9 (including active forms) expression in CA. These findings agree with the remodeling induced by blood flow reduction, where MMP activation has been observed (2, 24). Therefore, CBF attenuation induces collagenolytic activity by MMPs that will contribute to the MCA hypotrophy observed at this stage. The resulting remodeling elevates wall stress, which in turn will signal for the further development of hypertro-phy in an attempt to decrease wall tension. However, low-flow-induced remodeling independent of MMPs has also been described (15).

Alterations in vascular mechanical properties may take part in the cerebral disturbance caused by ischemic processes (23). Arterial stiffness is influenced by ECM proteins, and, in gen-eral, collagen contributes to enhanced stiffness (4). MCA from BCCAO rats showed diminished collagen I/III expression, and thus decreased stiffness would be expected. Instead, here wall stiffness remained unchanged after 15 days of permanent BCCAO. Similarly, paradoxical alterations of collagen and compliance have already been reported under other conditions (20, 43). Previous studies support that, besides collagen con-tent, collagen organization contributes to define the stiffness of the arterial wall (4, 21). Furthermore, not only collagen but

Fig. 6. Representative immunoblot and densitometric analysis of matrix metalloproteinase (MMP)-1 (A) and latent and active MMP-9 protein expression (B) in CA from sham and BCCAO rats. The expression of GAPDH as a loading control is also shown. Values are means⫾SE ofn⫽6 – 8 rats. *P⬍0.05; **P


also other ECM components (i.e., elastin) are involved in arterial stiffness and might counteract the possible effects of decreased collagen. Finally, we cannot exclude that collagen reduction was an early event preceding further alterations in stiffness at later time points.

Numerous studies have only attempted to analyze the influ-ence of vascular remodeling in several disease states. However, CBF is tightly regulated by myogenic mechanisms to maintain a relatively stable cerebral perfusion. Myogenic response as a function of pressure reveals the extent of the constrictor tone, whereas myogenic reactivity refers to vasoconstriction of the vessel to pressure once tone is present (10). In the present study, although myogenic reactivity was preserved, myogenic response was decreased at all of the perfusion pressures tested. It is well accepted that after a transient ischemic episode, one of the mechanisms involved in myogenic impairment of rat cerebral arteries is the increased peroxynitrite production caus-ing loss of F-actin (22, 28, 29). However, vascular myogenic properties are also intricately linked to changes in wall struc-ture. The vascular ECM is subject to tensile force produced by blood pressure and transferred through integrins. Among oth-ers, these integrins can form signaling complexes that regulate cytoskeletal dynamics, indirectly regulating myogenic capacity (30). ECM disruption (i.e., collagen degradation by MMP) might directly impact these signaling complexes, contributing to myogenic impairment. However, the relationship between arterial remodeling and myogenic autoregulation is still far from being well understood.

In a previous study, we showed a decrease in MCA myo-genic response from normotensive compared with hypertensive rats after transient MCA occlusion/reperfusion, contributing to an explanation of the smaller infarct volume in the former (23). Furthermore, we subsequently observed that reduction of the cerebral infarct size by an antioxidant treatment was not coupled to normalization of the MCA myogenic response (22). In those studies, including the present one, decreased myogenic response was accompanied by preserved myogenic reactivity. Together, these findings suggest that decreased MCA myo-genic response could be considered as an adaptive response to facilitate and/or preserve blood flow irrigation, thus limiting the damage in the surrounding tissue. Consistently, marked cortical neuronal damage is not normally observed at early stages, despite findings that behavioral decline is an early-emerging process already established after a few weeks of permanent BCCAO (39, 55). These changes are associated to white matter alterations early after BCCAO (36, 51, 55). Thus, after MCA occlusion, injury to the white matter occurs before histological changes within the cortex become apparent (38). Blood flow to the cortex is supplied by the cortical branches of the MCA, whereas the white matter is supplied by its pene-trating branches. For instance, these downstream arterioles are less susceptible to changes due to ischemia-reperfusion than upstream MCA, because the myogenic response of penetrating branches remains unaltered (11), suggesting they are also less able to adapt to ischemic insults. Therefore, the regions sup-plied by these perforating branches of the MCA could be more vulnerable than the cortex to cerebral hypoperfusion.

In conclusion, this study shows that chronic reduction of CBF induces hypotrophic remodeling of the MCA linked to decreased collagen I/III. The decreased MCA myogenic re-sponse could be considered as an adaptive rere-sponse to assure

correct tissue perfusion and may contribute to prevent early significant neuronal damage. These results help to understand the impact of reducing blood flow to the brain and the mech-anisms by which the cerebrovasculature adapts to prevent brain tissue damage.


We thank the confocal microscopy core from Universitat Autònoma de Barcelona.


This work was supported by Ministerio de Ciencia e Innovación Grant SAF 2007-60406 (to E. Vila) and Generalitat de Catalunya Grant 2009SGR-890. A. Márquez-Martín was supported by Universitat Autònoma de Barcelona. DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS

Author contributions: A.M.-M., A.P.D., and L.C. performed experiments; A.M.-M. and A.P.D. analyzed data; A.M.-M., F.J.-A., and E.V. interpreted results of experiments; A.M.-M. prepared figures; A.M.-M. and F.J.-A. drafted manuscript; F.J.-A., A.M.P., and E.V. edited and revised manuscript; A.M.P. and E.V. conception and design of research; E.V. approved final version of manuscript.


1. Aytac E, Seymen HO, Uzun H, Dikmen G, Altug T.Effect of iloprost on visual evoked potentials and brain tissue oxidative stress after bilateral common carotid artery occlusion. Prostaglandins Leukot Essent Fatty Acids74: 373–378, 2006.

2. Bassiouny HS, Song RH, Hong XF, Singh A, Kocharyan H, Glagov S. Flow regulation of 72-kD collagenase IV (MMP-2) after experimental arterial injury.Circulation98: 157–163, 1998.

3. Baumbach GL, Heistad DD.Remodeling of cerebral arterioles in chronic hypertension.Hypertension13: 968 –972, 1989.

4. Briones AM, Arribas SM, Salaices M.Role of extracellular matrix in vascular remodeling of hypertension.Curr Opin Nephrol Hypertens19: 187–194, 2010.

5. Buus CL, Pourageaud F, Fazzi GE, Janssen G, Mulvany MJ, De Mey JG. Smooth muscle cell changes during flow-related remodeling of rat mesenteric resistance arteries.Circ Res89: 180 –186, 2001.

6. Chan SL, Tabellion A, Bagrel D, Perrin-Sarrado C, Capdeville-Atkinson C, Capdeville-Atkinson J. Impact of chronic treatment with red wine polyphenols (RWP) on cerebral arterioles in the spontaneous hypertensive rat.J Cardiovasc Pharmacol51: 304 –310, 2008.

7. Choy M, Ganesan V, Thomas DL, Thornton JS, Proctor E, King MD, van der Weerd L, Gadian DG, Lythgoe MF.The chronic vascular and haemodynamic response after permanent bilateral common carotid occlu-sion in newborn and adult rats.J Cereb Blood Flow Metab26: 1066 –1075, 2006.

8. Cipolla MJ. The Cerebral Circulation. San Rafael, CA: Morgan & Claypool Life Sciences, 2009.

9. Cipolla MJ, De Lance N, Vitullo L.Pregnancy prevents hypertensive remodeling of cerebral arteries: a potential role in the development of eclampsia.Hypertension47: 619 –626, 2006.

10. Cipolla MJ, McCall AL, Lessov N, Porter JM.Reperfusion decreases myogenic reactivity and alters middle cerebral artery function after focal cerebral ischemia in rats.Stroke28: 176 –180, 1997.

11. Cipolla MJ, Smith J, Kohlmeyer MM, Godfrey JA.SKCa and IKCa Channels, myogenic tone, and vasodilator responses in middle cerebral and parenchymal arterioles: effect of ischemia and reperfusion.Stroke40: 1451–1457, 2009.

12. De Jong GI, Farkas E, Stienstra CM, Plass JRM, Keijser JN, de la Torre JC, Luiten PG.Cerebral hypoperfusion yields capillary damage in the hippocampal CA1 area that correlates with spatial memory impair-ment.Neuroscience91: 203–210, 1999.


13. de la Torre JC.Critically attained threshold of cerebral hypoperfusion: the CATCH hypothesis of Alzheimer’s pathogenesis.Neurobiol Aging21: 331–342, 2000.

14. de la Torre JC.Vascular risk factor detection and control may prevent Alzheimer’s disease.Ageing Res Rev9: 218 –225, 2010.

15. Dumont O, Loufrani L, Henrion D.Key role of the NO-pathway and matrix metalloproteinase-9 in high blood flow-induced remodeling of rat resistance arteries.Arterioscler Thromb Vasc Biol27: 317–324, 2007. 16. Farkas E, Luiten PG, Bari F.Permanent, bilateral common carotid artery

occlusion in the rat: a model for chronic cerebral hypoperfusion-relates neurodegenerative diseases.Brain Res Rev54: 162–180, 2007. 17. Folkow B.Structural, myogenic, humoral, and nervous factors controlling

peripheral resistance. In: Hypotensive Drugs, edited by Harington M. London: Pergamon, 1956.

18. Fox G, Gallacher D, Shevde S, Loftus J, Swayne G.Anatomic variation of the middle cerebral artery in the Sprague-Dawley rat. Stroke 24: 2087–2092, 1993.

19. Franceschi M, Alberoni M, Bressi S, Canal N, Comi G, Fazio F, Grassi F, Perani D, Volonté MA.Correlations between cognitive impairment, middle cerebral artery flow velocity and cortical glucose metabolism in the early phase of Alzheimer’s disease.Dementia6: 32–38, 1995.

20. Intengan HD, Deng LY, Li JS, Schiffrin EL.Mechanics and composi-tion of human subcutaneous resistance arteries in essential hypertension.

Hypertension33: 569 –574, 1999.

21. Intengan HD, Schiffrin EL.Vascular remodeling in hypertension: roles of apoptosis, inflammation, and fibrosis.Hypertension38: 581–587, 2001. 22. Jiménez-Altayó F, Caracuel L, Pérez-Asensio FJ, Martínez-Revelles S, Messeguer A, Planas AM, Vila E.Participation of oxidative stress on rat middle cerebral artery changes induced by focal cerebral ischemia: beneficial effects of 3,4-dihydro-6-hydroxy-methoxy-2,2-dimethyl-1(2H)-benzopyran (CR-6).J Pharmacol Exp Ther331: 429 –436, 2009. 23. Jiménez-Altayó F, Martín A, Rojas S, Justicia C, Briones AM,

Gi-raldo J, Planas AM, Vila E.Transient middle cerebral artery occlusion causes different structural, mechanical, and myogenic alterations in nor-motensive and hypertensive rats.Am J Physiol Heart Circ Physiol293: H628 –H635, 2007.

24. Johnson C, Galis ZS.Matrix metalloproteinase-2 and -9 differentially regulate smooth muscle cell migration and cell-mediated collagen orga-nization.Arterioscler Thromb Vasc Biol24: 54 –60, 2004.

25. Kántor O, Schmitz C, Feise J, Brasnkevic I, Korr H, Busto R, Ginsberg MD, Schmidt-Kastner R.Moderate loss of cerebellar Purkinje cells after chronic bilateral common carotid artery occlusion in rats.Acta Neuropathol (Berl)113: 549 –558, 2007.

26. Lambert CM, Roy M, Robitaille GA, Richard DE, Bonnet S.HIF-1 inhibition decreases systemic vascular remodeling diseases by promoting apoptosis through a hexokinase 2-dependent mechanism.Cardiovasc Res

88: 196 –204, 2010.

27. Loufrani L, Levy BI, Henrion D.Defect in microvascular adaptation to chronic changes in blood flow in mice lacking the gene encoding for dystrophin.Circ Res91: 1183–1189, 2002.

28. Maneen MJ, Cipolla MJ. Peroxynitrite diminishes myogenic tone in cerebral arteries: role of nitrotyrosine and F-actin.Am J Physiol Heart Circ Physiol292: H1042–H1050, 2007.

29. Maneen MJ, Hannah R, Vitullo L, DeLance N, Cipolla MJ. Peroxyni-trite diminishes myogenic activity and is associated with decreased vas-cular smooth muscle F-actin in rat posterior cerebral arteries.Stroke37: 894 –899, 2006.

30. Martinez-Lemus LA, Crow T, Davis MJ, Meininger GA. ␣v␤3- and ␣5␤1-integrin blockade inhibits myogenic constriction of skeletal muscle resistance arterioles.Am J Physiol Heart Circ Physiol289: H322–H329, 2005.

31. Martinez-Lemus LA, Hill MA, Meininger GA.The plastic nature of the vascular wall: a continuum of remodeling events contributing to control of arteriolar diameter and structure.Physiology (Bethesda)24: 45–57, 2009. 32. Mathiassen ON, Buus NH, Sihm I, Thybo NK, Mørn B, Schroeder AP, Thygesen K, Aalkjaer C, Lederballe O, Mulvany MJ, Christensen KL.Small artery structure is an independent predictor of cardiovascular events in essential hypertension.J Hypertens25: 1021–1026, 2007. 33. Mulvany MJ, Aalkjaer C. Structure and function of small arteries.

Physiol Rev70: 921–961, 1990.

34. Murata K, Motayama T, Kotake C.Collagen types in various layers of the human aorta and their changes with the atherosclerotic process.

Atherosclerosis60: 251–262, 1986.

35. Novensa L, Select J, Pastor M, Sandberg K, Heras M, Dantas AP. Equine 40 estrogens impair nitric oxide production and endothelial nitric oxide synthase transcription in 41 human endothelial cells compared with the natural 17␤-estradiol.Hypertension56: 405–411, 2010.

36. Otori T, Katsumata T, Maramatsu H, Kashiwagi F, Katayama Y, Terashi A.Long-term measurement of cerebral blow flow and metabo-lism in a rat chronic hypoperfusion model.Clin Exp Pharmacol Physiol

30: 266 –272, 2003.

37. Ozacmak VH, Sayan H, Catin A, Akyildiz-Igdem A.AT1 receptor blocker candersartan-induced attenuation of brain injury of rats subjected to chronic cerebral hypoperfusion.Neurochem Res32: 1314 –1321, 2007. 38. Pantoni L, Garcia JH, Gutierrez JA.Cerebral white matter is highly

vulnerable to ischemia.Stroke27: 1641–1647, 1996.

39. Pappas BA, de la Torre JC, Davidson CM, Keyes MT, Fortin T. Chronic reduction of cerebral blood flow in the adult rat: late-emerging CA1 cell loss and memory dysfunction.Brain Res708: 50 –58, 1996. 40. Pourageaud F, De Mey JG.Structural properties of rat mesenteric small

arteries after 4-wk exposure to elevated or reduced blood flow.Am J Physiol Heart Circ Physiol273: H1699 –H1706, 1997.

41. Raffetoo JD, Khalil RA.Matrix metalloproteinase and their inhibitors in vascular remodeling and vascular disease.Biochem Pharmacol75: 346 – 359, 2008.

42. Rizzoni D, Porteri E, Boari GE, De Ciuceis C, Sleiman I, Muiesan ML, Castellano M, Miclini M, Agabiti-Rosei E.Prognostic significance of small-artery structure in hypertension.Circulation108: 2230 –2235, 2003. 43. Sachidanandam K, Hutchinson JR, Elgebaly MM, Mezzetti EM, Dorrance AM, Motamed K, Ergul A.Glycemic control prevents micro-vascular remodeling and increased tone in type 2 diabetes: link to endo-thelin-1.Am J Physiol Regul Integr Comp Physiol296: R952–R959, 2009. 44. Schmidt-Kastner R, Aguirre-Chen C, Saul I, Yick L, Hamasaki D, Busto R, Ginsberg MD.Astrocytes react to oligemia in the forebrain induced by chronic bilateral common carotid artery occlusion in rats.

Brain Res1052: 28 –39, 2005.

45. Semenza GL.Expression of hypoxia-inducible factor 1: mechanisms and consequences.Biochem Pharmacol59: 47–53, 2000.

46. Semenza GL.Involvement of oxygen-sensing pathways in physiologic and pathologic erythropoiesis.Blood114: 2015–2019, 2009.

47. Stefani A, Sancesario G, Pierantozzi M, Leone G, Galati S, Hain-sworth AH, Diomedi M.CSF biomarkers, impairment of cerebral hemo-dynamics and degree of cognitive decline in Alzheimer’s and mixed dementia.J Neurol Sci283: 109 –115, 2009.

48. Tanaka K, Ogawa N, Asanuma M, Kondo S, Nomura M.Relationship between cholinergic dysfunction and discrimination learning disabilities in Wistar rats following chronic cerebral hypoperfusion. Brain Res 729: 55–65, 1996.

49. Tsuchiya M, Sako K, Yura S, Yonemasu Y.Cerebral blood flow and histopathological changes following permanent bilateral carotid artery ligation in Wistar rats.Exp Brain Res8: 87–92, 1991.

50. Villalba N, Martínez P, Bríones AM, Sánchez A, Salaíces M, García-Sacristán A, Hernández M, Benedito S, Prieto D.Differential structural and functional changes in penile and coronary arteries from obese Zucker rats.Am J Physiol Heart Circ Physiol297: H696 –H707, 2009. 51. Wakita H, Tomimoto H, Akiguchi I, Kimura J.Glial activation and

white matter changes in the rat brain induced by chronic cerebral hypo-perfusion: an immunohistochemical study.Acta Neuropathol (Berl)87: 484 –492, 1994.

52. Wakita H, Tomimoto H, Akiguchi I, Lin JX, Ihara M, Ohtani R, Shibata M.Ibudilast, a phosphodiesterase inhibitor, protects against white matter damage under chronic cerebral hypoperfusion in the rat.Brain Res

992: 53–59, 2003.

53. Wang J, Zhang HY, Tang XC.Huperzine A improves chronic inflam-mation and cognitive decline in rats with cerebral hypoperfusion. J Neurosci Res88: 807–815, 2010.

54. Yang YM, Feng X, Yao ZW, Tang WJ, Liu HQ, Zhang L.Magnetic resonance angiography of carotid and cerebral arterial occlusion in rats using a clinical scanner.J Neurosci Methods167: 176 –183, 2008. 55. Yanpallewar S, Rai S, Kumar M, Chauhan S, Acharya SB.

Neuropro-tective effect ofAzadirachta indicaon cerebral post-ischemic reperfusion and hypoperfusion in rats.Life Sci76: 1325–1338, 2005.





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