(Stroke. 2001;32:753.)
© 2001 American Heart Association, Inc.
Original Contributions |
Acute-Phase Proteins Before Cerebral Ischemia in Stroke-Prone Rats
Identification by Proteomics
From the Dipartimento di Scienze Farmacologiche, Facoltà di Farmacia, Università degli Studi, Milan, Italy (L.S., E.T., U.G., A.M.C., I.E., M.A., R.P., E.G.), and Institut für Medizinische Chemie, Veterinärmedizinische Universität, Vienna, Austria (I.M., M.G.).
Correspondence to Elisabetta Gianazza, PhD, Dipartimento di Scienze Farmacologiche, Facoltà di Farmacia, Università degli Studi di Milano, via Balzaretti 9, I-20133 Milan, Italy. E-mail Elisabetta.Gianazza{at}unimi.it
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Background and Purpose—A high degree of proteinuria has been reported in stroke-prone spontaneously hypertensive rats (SHRSP). We studied the effect of salt loading on the detailed protein pattern of serum and urine in 3 rat strains: Wistar-Kyoto, spontaneously hypertensive rats, and SHRSP, an inbred animal model for a complex form of cerebrovascular disorder resembling the human disease.
Methods—Rats were given a permissive diet and received 1% NaCl in drinking water. The protein pattern in body fluids was assessed over time by 2-dimensional electrophoretic analysis. Brain alterations were monitored by MRI and histology.
Results—Several
proteins were excreted in urine after weeks of treatment and in advance
of stroke: transferrin, hemopexin, albumin,
2-HS-glycoprotein,
kallikrein-binding protein, 1-antitrypsin,
Gc-globulin, and transthyretin. Markers of an inflammatory response,
including very high levels of thiostatin, were detected in the serum of
SHRSP at least 4 weeks before a stroke
occurred.
Conclusions—In SHRSP subjected to salt loading, an atypical inflammatory condition and widespread alterations of vascular permeability developed before the appearance of anomalous features in the brain detected by MRI. Urinary concentrations of each of the excreted serum proteins correlated positively with time before stroke occurred.
Key Words: animal models • inflammation • nuclear magnetic resonance • proteome • stroke • rats
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The spontaneously hypertensive stroke-prone rat (SHRSP) provides an inbred animal model for a complex form of cerebrovascular pathology resembling, in many aspects, the human disease.1 In these animals stroke occurs only after high blood pressure has developed; lag time is greatly reduced if rats are exposed to a specific permissive diet, low in potassium and protein and high in sodium (Japanese permissive diet [JPD]).2 In this model, stroke is not just a consequence of long-standing hypertension but represents the final outcome of a complex interaction between environmental (ie, dietary) and genetic factors.3 The role of the latter is emphasized by the resistance to stroke of a closely related strain, the spontaneously hypertensive rat (SHR), which remains stroke-free despite a similar degree of hypertension after exposure to JPD.
Recent data show that alterations in structure, regulation, and function of the gene encoding atrial natriuretic peptide (ANP) are significantly linked to cerebral disease in SHRSP.4 Further reports suggest that molecular variants of the ANP gene represent an independent risk factor for human stroke.5 These data confirm the resemblance between cerebrovascular accidents in humans and in the SHRSP model.
It has been shown that cerebral edema identified by T2-weighted MRI in salt-loaded SHRSP is preceded by proteinuria.6 Preliminary experiments performed on urine samples of SHRSP showed that, in addition to albumin, several acute-phase proteins are detectable by means of 2-dimensional electrophoresis. This observation suggested to us that in this animal model inflammation might occur well before the onset of the acute cerebral event. Therefore, this study addressed the issue of a potential contribution of acute inflammation to the occurrence of cerebral damage in salt-loaded SHRSP.
The term proteome refers to proteins expressed by a genome in a given tissue at a given time; its analysis relies on 2-dimensional polyacrylamide gel electrophoresis. The simultaneous characterization and quantification in body fluids of gene products and of their posttranslational modifications can suggest which biochemical mechanisms are involved in a pathological situation and may identify disease markers.7 8 Our group has extensively studied the proteome of rat serum, identifying and quantifying all major proteins under baseline conditions and after a number of experimental treatments.9 10 11 12 13 14
In this report the proteome of serum and urine in SHRSP rats exposed to JPD during and after the development of cerebral damage, in comparison with SHR and Wistar-Kyoto rats (WKY), was characterized by 2-dimensional polyacrylamide gel electrophoresis.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Animals and Treatments
Procedures involving animals and their care were conducted at Dipartimento di Scienze Farmacologiche dell’Università degli Studi di Milano in conformity with the institution’s guidelines, which are in compliance with national (D.L. No. 116, G.U., suppl. 40, February 18, 1992, Circolare No. 8, G.U., July 14, 1994) and international (EEC Council Directive 86/609, OJL 358, 1, December 12, 1987; Guide for the Care and Use of Laboratory Animals, US National Research Council, 1996) rules and policies.
Male WKY (n=5), SHR (n=5), and SHRSP (n=8) were purchased from Charles River. At 6 weeks of age the animals were placed on JPD (Laboratorio Dr Piccioni; 18.7% protein, 0.63% potassium, 0.37% sodium) and received 1% NaCl in drinking water.
Animal treatment lasted up to 49 days. The body weight of the animals, as well as food and liquid intake, was recorded weekly. Serum and 24-hour urine were sampled and arterial blood pressure was monitored before the onset of the diet and every seventh day afterward. Systolic arterial blood pressure was measured in conscious rats by tail-cuff plethysmography (PB Recorder 8006, Ugo Basile), after warming at 37°C.
Blood was drawn from the tail vein at different time intervals (see Results), and serum was obtained by allowing the blood to clot for 1 hour at 37°C followed by centrifugation for 20 minutes at 3000 rpm. Urine was collected by housing rats in individual metabolic cages; its protein concentration was measured according to Bradford15 with bovine albumin as standard.
All rats underwent weekly MRI. MRI assessment was repeated every other day in SHRSP once 24-hour proteinuria exceeded 40 mg/d6 and daily after brain abnormality had been detected by T2-weighted MRI.
MRI Evaluations
Rats were anesthetized with 2% isoflurane in
70% N2/30% O2, fixed on
the animal holder by a rod beneath the teeth, and placed into the
magnet (4.7 T, vertical 15-cm bore) of a Bruker spectrometer (AMX3 with
microimaging accessory). A 6.4-cm-diameter birdcage coil was used for
imaging. After a 3-orthogonal plane gradient echo scout, a T2
multislice image was obtained. Sixteen contiguous 1-mm-thick slices
were analyzed caudal to the olfactory bulb; field of view was
4x4 cm2. Turbo spin-echo sequence was used
with 16 echoes per excitation, 10 ms interecho time, 85 ms equivalent
echo time, and 4 seconds repetition time. The images were 128x128
points (zero filled to 256x256); 8 images were averaged in 8 minutes
and 30 seconds. The occurrence of lesions was identified with presence
of areas of high signal intensity on T2-weighted MRI. The extension of
the lesions was determined by thresholding the images and interactively
drawing outlines of the lesion and of the whole brain; the ratio
between the number of pixels in the lesion versus that in the whole
brain in each slice gave the percentage of the brain affected by the
lesion. The percentage of hyperintense pixels was evaluated in 9
slices: 4 caudal and 4 rostral to the central slice where the primary
lesion had been detected.
Histology
After the last MRI session, the anesthetized
rats were perfused through the left ventricle with 4%
paraformaldehyde in 0.1 mmol/L phosphate buffer.
The brains were dissected, postfixed overnight in the same solution,
cryoprotected by immersion in 30% sucrose-phosphate buffer, and
sectioned with a cryotome. Coronal sections, 10 µm thick, were
stained with hematoxylin-eosin and/or toluidine blue and examined
microscopically.
Electrophoretic Techniques
One-dimensional electrophoresis was mainly
used to assess the relative concentrations of the major components
(transferrin and albumin in serum, -2u-globulin in urine),
and 2-dimensional electrophoresis was mainly used to evaluate with high
sensitivity the average- and low-abundance components under conditions
in which the signal for albumin was saturated.
One-dimensional electrophoresis was run in the presence of SDS, without sample reduction, in a discontinuous buffer system16 on polyacrylamide gradients 4% to 20% T. The sample load was 3.75 µg of urine proteins and 0.125 µL of serum per lane. Two-dimensional electrophoresis maps were obtained by the immobilized pH gradient (IPG)–Dalt method.17 Sample proteins, reduced with 2% 2-mercaptoethanol, were first resolved according to charge on a nonlinear pH 4 to 10 IPG18 in the presence of 8 mol/L urea and 0.5% carrier ampholytes, with an anode-to-cathode distance of 8 cm. The focused proteins were then fractionated according to size by SDS-PAGE on 7.5% to 17.5% polyacrylamide gradients, with 2 IPG strips mounted on each 160x140-mm2 SDS slab. Sample loads were 100 µg of urine proteins or 2 µL of serum. Proteins were stained with 0.3% wt/vol Coomassie or with silver nitrate19 for some urine samples.
The protein patterns were scanned with a video camera under the control of NIH Image, release 1.61, and analyzed with the software PDQUEST version 5.1 (PDI).20 Data for individual proteins (spots or spot chains identified by immunological or physicochemical means9 10 11 ) are reported as spot volumes as a function of time before or after MRI evidence of stroke.
Statistical Analysis
Linear and nonlinear fits were taken as statistically
significant for P<0.05. For
every fit, R and
P values are listed in the
Table
.
Differences between groups were evaluated by ANOVA for repeated
measurements, followed by Bonferroni’s post hoc test. For SHRSP this
analysis was limited to day 42 from the start of the treatment,
the last time point at which all these rats still
survived.
|
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Physical Parameters
The growth of SHRSP was slower than that of SHR and WKY. Between days 28 and 49 of treatment, SHRSP lost 10% of body mass and weighed 55% to 65% of the age-matched controls at the time of euthanasia (Figure 1a
).
|
At baseline, WKY, SHR, and SHRSP had systolic blood
pressure of 118±5, 120±10, and 130±8 mm Hg, respectively.
Systolic blood pressure of SHR and SHRSP, but not of WKY,
increased markedly during JPD and salt loading
(Figure 1b
).
Identification of Brain Damage by MRI and
Histological Analysis
As a result of salt loading, all SHRSP developed
cerebral lesions, at a median of 42±3 days from the start of
treatment. Brain lesions occurred within the gray matter, usually in
the frontal or forelimb areas of cortex or in the ventral region of the
caudate putamen
(Figure 2a). From these single foci the lesions then spread
throughout the brain. In 3 of 8 rats the damage reached the
contralateral hemisphere via the corpus callosum (not shown). Three
days after the first evidence of brain damage, the extent of the lesion
averaged 12.5±1.6% (SD; n=8).
|
Histological evaluation confirmed that the
alterations in brain MRI identified areas characterized by tissue
thinning due to loss of neuronal cells
(Figure 2b). Neither SHR nor WKY developed brain
abnormalities detectable with MRI or histological
examination throughout the experimental period (not
shown).
Proteomics Data
Urine
Between days 0 and 7 of salt loading, protein loss per
day (24-hour proteinuria) increased 3- to 4-fold in all experimental
groups, but thereafter it changed little for WKY and SHR
(Figure 3a). In SHRSP, mean protein loss increased sharply
(and linearly) after day 28 of treatment
(Figure 3). The delay between the time when proteinuria
(evaluated once a week) indicated a protein loss 
40
mg/d6 and the time when MRI,
performed twice a week, identified focal cerebral ischemia was
9±3 days. In 6 of 8 rats, urine protein loss peaked immediately before
stroke and declined thereafter.
|
In SHRSP, not only the total amount of protein in urine but
also the qualitative protein composition changed with time, as shown by
1-dimensional electrophoretic analysis through SDS-PAGE
(Figure 4). The ratio between 20-kDa components, ie,
-2u-globulin, and 65- to 68-kDa components, mostly albumin,
dropped between days 28 and 35 of treatment from approximately 15
(typical for WKY) to approximately 0
(Figure 4a). The largest protein found in SHRSP urine was
transferrin (molecular weight [Mr], 77 kDa).
The ratio of transferrin to 65- to 68-kDa components in urine increased
from 0 (typical of WKY) to approximately 0.2, with most of these
changes occurring between days 28 and 35 of treatment. After day 35,
both ratios in SHRSP differed significantly from those in WKY (and SHR;
not shown). The ratio between 77-kDa and 65- to 68-kDa proteins in the
serum of rats did not change during the study period
(Figure 4b). At day 42, this ratio in urine of SHRSP had
approximated that in serum (0.18±0.03)
(Figure 4b and 4c) and had even surpassed it (0.20±0.02), so
that the bulky proteins were now leaking freely into the
urine.
|
Analysis of the protein pattern in urine by
2-dimensional electrophoresis
(Figure 5) confirmed that -2u-globulin is by far
the major protein in male rat urine under baseline conditions.
Albumin and IgL were the only serum proteins recognized in the
pattern of WKY and SHR at all time
points,21 22 23
whereas in SHRSP the proteins of high Mr lost in
urine during treatment were resolved by 2-dimensional electrophoretic
analysis as transferrin, albumin, thiostatin,
kallikrein-binding protein,
2-HS-glycoprotein, serine
protease inhibitor 3,
1-antitrypsin, Gc-globulin, apolipoprotein
A-IV, transthyretin, and the albumin fragments migrating as
slanted rows with Mr 50 and 30 kDa. Hemopexin is
not easily recognized in Coomassie-stained patterns, but its presence
was confirmed by immunoblotting (not
shown).
|
Figure 6 shows the best-fit curves for individual SHRSP data
of the amounts of major proteins in urine and plasma versus time from
stroke. For every protein detected in urine, a positive and highly
significant linear correlation
(R0.67,
P<0.0001) was observed with
time
(Table).
The intercept of the regression line on the time axis was approximately
day -20 from stroke for most proteins in urine but corresponded to
-17 for thiostatin (versus -30 for its first appearance in serum)
and to -25 for transthyretin.
|
An extensive preliminary investigation of which gels were heavily loaded and silver-stained for high sensitivity detected no difference in the amounts of minor components among urine from WKY, SHR, and SHRSP until loss of serum proteins began in the latter (not shown).
Serum
The 2-dimensional electrophoretic patterns of WKY and
SHRSP sera after weeks of salt loading are shown in
Figure 5. Several qualitative and quantitative differences
were observed, most notably the appearance of thiostatin and serine
protease inhibitor 3 and the drastic decrease of
2-HS-glycoprotein, transferrin,
and albumin. The long-term course for major proteins is plotted
in
Figure 6 as a function of time from stroke. For 5 proteins
(apolipoprotein A-IV, transthyretin, apolipoprotein E, retinol-binding
protein, 1-inhibitor III; not
shown) no definite trend was detected; the increase in group-specific
component levels over time was very low. The concentrations of
thiostatin and 1-antitrypsin+serine protease
inhibitor 3 increased linearly with time; those of
transferrin, kallikrein-binding
protein+2-HS-glycoprotein,
haptoglobin, and C-reactive protein varied according to a second-order
curve, with maximal values in all cases at approximately day -20
before stroke.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In the present report we describe the occurrence of inflammatory markers in urine and plasma in an experimental model, SHRSP, that develops brain damage invariably preceded by massive proteinuria.
Before the occurrence of ischemic damage, the serum
protein pattern of SHRSP resembles that of experimental
inflammation.10 11 12 13
Specifically, 1-antitrypsin+serine protease
inhibitor 3 and thiostatin increase linearly in serum
throughout the duration of the treatment. Kallikrein-binding
protein+2-HS-glycoprotein
increase at first, then decrease, with
2-HS-glycoprotein, a negative
acute-phase protein, almost vanishing at the latest experimental times
(Figure 5).
In humans, C-reactive protein is the most sensitive marker
of many inflammatory conditions, since its level increases by 1 order
of magnitude within a few hours from the noxious
stimulus,24 and several
studies have identified C-reactive protein as a strong predictor of
myocardial
infarction25 26
and stroke.27 It should be
noted, however, that the set of proteins affected in acute-phase
reactions differs depending on the
species.28 29 In
the rat, C-reactive protein levels are only marginally affected during
early inflammation; serum amyloid A is lacking altogether from rat
serum. On the contrary, in the rat a dramatic rise in thiostatin (also
called 1-major acute-phase protein), a
species-specific thiol protease
inhibitor,30 31
as well as an increase in 2-macroglobulin are
observed during inflammation. In SHRSP most of the acute-phase proteins
begin to increase over basal levels soon after the start of the
treatment
(Figure 6); thiostatin is first detected at least 4 weeks
before the occurrence of stroke.
Most of the acute-phase proteins found in serum are also
detected in urine of SHRSP, and their level increases over time, with
maximal values at the date of euthanasia
(Figure 6). A time delay with respect to serum that varies
from one protein to another is observed. In particular, transthyretin
(Mr, 56 kDa) is detected in urine approximately
25 days before the appearance of ischemic brain features;
thiostatin is detected only 16 days before. Thus, selectivity of
excretion in terms of both size and charge is noticed. Indeed, the
higher 77 kDa/65 to 68 kDa ratio measured in urine than in serum
(Figure 4) is due to preferential loss of albumin
versus other components of similar Mr
(Figure 5). In addition, in urine samples from SHRSP only the
most alkaline isoforms (glycoforms) of transferrin are observed
(Figure 5).
-2u-Globulin32 is a
pheromone-binding lipocalin synthesized in male rat liver under the
influence of androgens and readily lost in urine. Its excretion is
known to be reduced in animals on a low-protein
diet.33 After 42 days of
treatment, SHRSP excrete <7 mg/d -2u-globulin versus 12 mg/d for
both WKY and SHR.
An increase in proteinuria before the occurrence of stroke in susceptible animals (SHRSP) subjected to high sodium intake has been previously reported.6 No information, however, was available on the protein composition of urine of SHRSP compared with WKY or SHR. Our data detail which proteins are lost in urine, as well as the time course of their excretion, in relationship to their varying concentration in serum.
The changes in the serum levels of several acute-phase proteins, with some of the positive acute-phase proteins being eventually excreted in urine, point to an inflammatory condition developing before stroke. In the present investigation we have not collected evidence on the tissues where the inflammatory status first develops in SHRSP. Data in the literature report extensive kidney abnormalities in this animal model, including thrombotic, proliferative, and necrotic lesions in arterioles and glomeruli,34 35 36 often preceding the occurrence of stroke.1 37 38 In addition, for patients with end-stage renal disease, a complex and mutual interaction between acute-phase inflammatory process and all aspects of kidney disease (uremia, heart failure, malnutrition, anemia) has been proposed.39 Since subpopulations of individuals with high levels of C-reactive protein and cytokines have been reported among patients both with normal renal function and with chronic renal failure, no conclusion is possible about cause versus consequence.39 Definitely, genetic background plays a relevant role in SHRSP because neither organ failure nor acute-phase reaction is observed in the related rat strain SHR under identical environment conditions.
Treatment with angiotensin I–converting enzyme inhibitors and angiotensin II antagonists prevents renal disorders, including proteinuria, as well as the occurrence of brain lesions.40 41 42 43 The protective effects of these drugs, however, are independent of alterations in blood pressure, which supports the hypothesis that development of vascular injury requires the concurrent involvement of local factors. In this experimental model the same drugs also exert protective effects against cerebral ischemia, which in turn suggests a potential role in the control of vascular permeability as well as of the development of an inflammatory reaction.
An increasing number of in vivo data suggest that the inflammatory response that follows stroke is involved in the pathogenesis of cerebral ischemia.44 Cytokines, synthesized locally during the development of brain lesions, have been suggested to exacerbate brain ischemic injury by several mechanisms; they activate the synthesis of acute-phase reactants.45 Less is known about the prognostic value of inflammatory markers in this pathological condition. C-reactive protein levels, however, have been reported to be an independent predictor not only of cardiovascular events but also of ischemic brain disease.26 46 Moreover, C-reactive protein concentration within 72 hours of an ischemic stroke is either an independent predictor of survival27 or an irrelevant parameter.47 Thus, an inflammatory reaction may also have a pathogenetic role in the occurrence of acute stroke. Experimental models to test this hypothesis, however, are lacking. SHRSP exposed to JPD, in which inflammation develops before the appearance of anomalous features in brain MRI, represent a useful experimental tool to understand how inflammation contributes to ischemic brain injury and to answer the question of whether anti-inflammatory strategies may affect genesis, progression, and outcome of brain damage. On the basis of this growing understanding, new pharmacological approaches should be designed that will enlarge our therapeutic armamentarium for the prevention and/or treatment of brain disease.
|
Acknowledgments |
|---|
This study was supported in part by a grant from Università degli Studi di Milano (Fondi ex-60%) to Dr Gianazza. The authors gratefully acknowledge expert advice on histological procedures from Professor Mauro Cimino. They are also very thankful to Silvia Castiglioni for assistance with analytical techniques and to Lina Belcastro for copying data into our database.
Received September 6, 2000; revision received November 27, 2000; accepted November 28, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Okamoto K, Yamori Y, Nagaoka A. Establishment of the stroke-prone spontaneously hypertensive rats (SHR). Circ Res. 1974;33/34:I-143–I-153.
2.
Yamori Y, Horie R,
Tanase H, Fujiwara K, Nara Y, Lovenberg W. Possible role of nutritional
factors in the incidence of cerebral lesions in stroke-prone
spontaneously hypertensive rats.
Hypertension. 1984;6:49–53.
3. Volpe M, Russo R, Vecchione C, Savoia C, Piras O, Gigante B, Lindpaintner K, Rubattu S. Endothelial dysfunction and genetic predisposition to stroke in spontaneously hypertensive rats. J Hypertens. 1998;16(suppl 8):S31–S35.
4.
Rubattu S,
Lee-Kirsh MA, DePaolis P, Giliberti R, Gigante B, Lombardi A, Volpe M,
Lindpaintner K. Altered structure, regulation, and function of the gene
encoding the atrial natriuretic peptide in the stroke-prone
spontaneously hypertensive rat. Circ
Res. 1998;85:900–905.
5.
Rubattu S,
Ridker P, Stampfer MJ, Volpe M, Hennekens CH, Lindpaintner K. The gene
encoding atrial natriuretic peptide and the risk of human
stroke. Circulation. 1999;100:1722–1726.
6.
Blezer ELA,
Schurink M, Nicolay K, Bär D, Jansen GH, Koomans HA, Joles JA.
Proteinuria precedes cerebral edema in stroke-prone rats: a magnetic
resonance imaging study.
Stroke. 1998;29:167–174.
7. Hochstrasser DF. Proteome in perspective. Clin Chem Lab Med. 1998;36:825–836.[Medline] [Order article via Infotrieve]
8. Blackstock WP, Weir MP. Proteomics: quantitative and physical mapping of cellular proteins. Trends Biotechnol. 1999;17:121–127.[Medline] [Order article via Infotrieve]
9. Haynes P, Miller I, Aebersold R, Gemeiner M, Eberini I, Lovati MR, Manzoni C, Vignati M, Gianazza E. Proteins of rat serum, I: establishing a reference 2-DE map by immunodetection and microbore high performance liquid chromatography–electrospray mass spectrometry. Electrophoresis. 1998;19:1484–1492.[Medline] [Order article via Infotrieve]
10. Miller I, Haynes P, Gemeiner M, Aebersold R, Manzoni C, Lovati MR, Vignati M, Eberini I, Gianazza E. Proteins of rat serum, II: influence of some biological parameters on the 2-DE pattern. Electrophoresis. 1998;19:1493–1500.[Medline] [Order article via Infotrieve]
11. Miller I, Haynes P, Eberini I, Gemeiner M, Aebersold R, Gianazza E. Proteins of rat serum, III: gender-related differences in protein concentration under baseline conditions and upon experimental inflammation. Electrophoresis. 1999;20:836–845.[Medline] [Order article via Infotrieve]
12. Eberini I, Miller I, Zancan V, Bolego C, Puglisi L, Gemeiner M, Gianazza E. Proteins of rat serum, IV: time-course of acute phase protein expression and its modulation by indomethacin. Electrophoresis. 1999;20:846–853.[Medline] [Order article via Infotrieve]
13. Eberini I, Agnello D, Miller I, Villa P, Fratelli M, Ghezzi P, Gemeiner M, Chan JH, Aebersold R, Gianazza E. Proteins of rat serum, V: adjuvant arthritis and its modulation by nonsteroidal antiinflammatory drugs. Electrophoresis. 2000;21:2170–2179.[Medline] [Order article via Infotrieve]
14. Eberini I, Miller I, Gemeiner G, Haynes P, Aebersold R, Puglisi L, Sirtori CR, Gianazza E. A Web site for the Rat Serum Protein Study Group. Electrophoresis. 1999;20:3599–3602.[Medline] [Order article via Infotrieve]
15. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of proteins utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–254.[Medline] [Order article via Infotrieve]
16. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680–685.[Medline] [Order article via Infotrieve]
17. Gianazza E. Casting immobilized pH gradients. In: Link AJ, ed. 2-D Proteome Analysis Protocols: Methods in Molecular Biology. Totowa, NJ: Humana Press; 1998.
18. Gianazza E, Giacon P, Sahlin B, Righetti PG. Non-linear pH courses with immobilized pH gradients. Electrophoresis. 1985;6:53–56.
19. Heukeshoven J, Dernick R. Neue Ergebnisse zum Mechanismus der Silberfärbung. In: Radola BJ, ed. Elektrophorese Forum ‘86. Weinheim, Germany: VCH; 1986:22–27.
20. Garrels JI. The QUEST system for quantitative analysis of two-dimensional gels. J Biol Chem. 1989;25:5269–5282.
21.
Alt JM, Hackbarth
H, Deerberg F, Stolte H. Proteinuria in rats in relation to
age-dependant renal changes. Lab
Anim. 1980;14:95–101.
22. Feld LG, Springate JE, Van Liew JB. Age-related changes in serum proteins of the spontaneously hypertensive rat. Proc Soc Exp Biol Med. 1988;188:480–484.[Medline] [Order article via Infotrieve]
23.
Marshall T,
Williams KM, Bayard C, Vesterberg O. Electrophoresis and silver
staining of rat urinary proteins including -2u-globulin.
Appl Theor Electrophor. 1992;2:189–192.[Medline]
[Order article via Infotrieve]
24. Pepys MB, Baltz ML. Acute phase proteins with special reference to C-reactive protein and related proteins (pentaxins) and serum amyloid A protein. Adv Immunol. 1983;34:141–212.[Medline] [Order article via Infotrieve]
25. Pietila KO, Harmoinen AP, Jokiniitty J, Pasternack AI. Serum C-reactive protein concentration in acute myocardial infarction and its relationship to mortality during 24 months of follow-up in patients under thrombolytic treatment. Eur Heart J. 1997;17:1345–1349.
26.
Ridker PM,
Hennekens CH, Buring JE, Rifai N. C-reactive protein and other markers
of inflammation in the prediction of cardiovascular
disease in women. N Engl J
Med. 2000;342:836–843.
27.
Muir KW, Weir CJ,
Alwan W, Squire IB, Lees KR. C-reactive protein and outcome after
ischemic stroke.
Stroke. 1999;30:981–985.
28.
Schreiber G,
Howlett G, Nagashima M, Millership A, Martin H, Urban J, Kotler L. The
acute phase response of plasma protein synthesis during experimental
inflammation. J Biol Chem. 1982;257:10271–10277.
29. Kushner I. Induction and control of acute phase reactants synthesis. In: Arnaud P, Benvenu J, Laurent P, eds. Marker Proteins in Inflammation. Vol 2. Berlin, Germany: W deGruyter; 1984:3–14.
30.
Urban J, Chan D,
Schreiber G. A rat serum glycoprotein whose synthesis rate
increases greatly during inflammation.
J Biol Chem. 1979;254:10565–10568.
31.
Cole T, Inglis
AS, Roxburgh CM, Howlett GJ. Major acute phase
1-protein of the rat is homologous to bovine
kininogen and contains the sequence of bradykinin: its synthesis is
regulated at the mRNA level. FEBS
Lett. 1985;182:57–61.[Medline]
[Order article via Infotrieve]
32.
Ichiyoshi Y, Endo
H, Yamamoto M. Length polymorphism in the 3' noncoding region of
rat hepatic -2u-globulin mRNAs. Biochim
Biophys Acta. 1987;910:43–51.[Medline]
[Order article via Infotrieve]
33.
Neuhaus OW, Flory
W. The effect of dietary protein on the excretion of
2u, the sex-dependent protein of the adult
male rat. Biochim Biophys Acta. 1975;411:74–86.[Medline]
[Order article via Infotrieve]
34.
Zuckermann A,
Chander PN, Zeballos GA, Stier CTJ. Regional nitric oxide release in
stroke-prone spontaneously hypertensive rats.
Hypertension. 1997;30:1479–1486.
35.
Blezer ELA,
Nicolay K, Goldschmeding R, Jansen GH, Koomans HA, Rabelink TJ, Joles
JA. Early-onset but not late-onset endothelin-A-receptor blockade can
modulate hypertension, cerebral edema, and proteinuria in stroke-prone
hypertensive rats.
Hypertension. 1999;33:137–144.
36.
Rocha R, Chander
PN, Zuckerman A, Stier CTJ. Role of aldosterone in renal
vascular injury in stroke-prone hypertensive rats.
Hypertension. 1999;33:232–237.
37. Nagaoka A, Iwatsuka H, Suzuoki Z, Okamoto K. Genetic predisposition to stroke in spontaneously hypertensive rats. Am J Physiol. 1976;230:1354–1359.
38. Shibota M, Nagaoka A, Shino A, Fujita T. Renin-angiotensin system in stroke-prone spontaneously hypertensive rats. Am J Physiol. 1979;236:H409–H416.
39. Lacson EJ, Owen WJ, Lowrie EG. What are the causes and consequences of the chronic inflammatory state in chronic dialysis patients? Semin Dial. 2000;13:164–166.
40.
Stier CTJ, Benter
IF, Ahmad S, Zuo HL, Selig N, Roethel S, Levine S, Itskovitz HD.
Enalapril prevents stroke and kidney dysfunction in salt-loaded
stroke-prone spontaneously hypertensive rats.
Hypertension. 1989;13:115–121.
41. Formes P, Richer C, Vacher E, Brunewal P, Giudicelli JF. Losartan’s protective effects in stroke-prone spontaneously hypertensive rats persist durably after treatment withdrawal. J Cardiovasc Pharmacol. 1993;22:305–313.[Medline] [Order article via Infotrieve]
42.
Blezer ELA,
Nicolay K, Bär D, Goldschmeding R, Jansen GH, Koomans HA, Joles JA.
Enalapril prevents imminent and reduces manifest cerebral edema in
stroke-prone hypertensive rats.
Stroke. 1998;29:1671–1678.
43. Hayashi K, Koyama M, Kido H, Egi Y, Kubo Y, Shinyama H, Iwamoto M, Nakamura N, Kagitani Y. Preventive and therapeutic effects of AE0047 on renal injury in stroke-prone spontaneously hypertensive rats. Clin Exp Pharmacol Physiol. 1997;24:831–840.[Medline] [Order article via Infotrieve]
44. Barone FC, Feuerstein GZ. Inflammatory mediators and stroke: new opportunities for novel therapeutics. J Cereb Blood Flow Metab. 1999;19:819–834.[Medline] [Order article via Infotrieve]
45. Vila N, Filella X, Deulofeu R, Ascaso C, Abellana R, Chamorro A. Cytokine-induced inflammation and long-term stroke functional outcome. J Neurol Sci. 1999;162:185–188.[Medline] [Order article via Infotrieve]
46.
Ridker PM,
Cushman M, Stampfer MJ, Tracy RP, Hennekens CH. Plasma concentration of
C-reactive protein and risk of developing peripheral
vascular disease. Circulation. 1998;97:425–428.
47. Canova CR, Courtin C, Reinhart WH. C-reactive protein (CRP) in cerebro-vascular events. Atherosclerosis. 1999;147:49–53. [Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  A. Gianella, E. Nobili, M. Abbate, C. Zoja, P. Gelosa, L. Mussoni, S. Bellosta, M. Canavesi, D. Rottoli, U. Guerrini, et al. Rosuvastatin Treatment Prevents Progressive Kidney Inflammation and Fibrosis in Stroke-Prone Rats Am. J. Pathol., April 1, 2007; 170(4): 1165 - 1177. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. H. Kobeissy, A. K. Ottens, Z. Zhang, M. C. Liu, N. D. Denslow, J. R. Dave, F. C. Tortella, R. L. Hayes, and K. K. W. Wang Novel Differential Neuroproteomics Analysis of Traumatic Brain Injury in Rats Mol. Cell. Proteomics, October 1, 2006; 5(10): 1887 - 1898. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Sironi, E. Gianazza, P. Gelosa, U. Guerrini, E. Nobili, A. Gianella, B. Cremonesi, R. Paoletti, and E. Tremoli Rosuvastatin, but not Simvastatin, Provides End-Organ Protection in Stroke-Prone Rats by Antiinflammatory Effects Arterioscler Thromb Vasc Biol, March 1, 2005; 25(3): 598 - 603. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Sironi, P. Gelosa, U. Guerrini, C. Banfi, V. Crippa, M. Brioschi, E. Gianazza, E. Nobili, A. Gianella, M. de Gasparo, et al. Anti-Inflammatory Effects of AT1 Receptor Blockade Provide End-Organ Protection in Stroke-Prone Rats Independently from Blood Pressure Fall J. Pharmacol. Exp. Ther., December 1, 2004; 311(3): 989 - 995. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Banfi, L. Sironi, G. De Simoni, P. Gelosa, S. Barcella, C. Perego, E. Gianazza, U. Guerrini, E. Tremoli, and L. Mussoni Pentoxifylline Prevents Spontaneous Brain Ischemia in Stroke-Prone Rats J. Pharmacol. Exp. Ther., September 1, 2004; 310(3): 890 - 895. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Maruvada and S. Srivastava Biomarkers for Cancer Diagnosis: Implications for Nutritional Research J. Nutr., June 1, 2004; 134(6): 1640S - 1645S. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Zabel, D. C. Chamrad, J. Priller, B. Woodman, H. E. Meyer, G. P. Bates, and J. Klose Alterations in the Mouse and Human Proteome Caused by Huntington's Disease Mol. Cell. Proteomics, May 1, 2002; 1(5): 366 - 375. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 U. Guerrini, L. Sironi, E. Tremoli, M. Cimino, B. Pollo, A. M. Calvio, R. Paoletti, and M. Asdente New Insights Into Brain Damage in Stroke-Prone Rats: A Nuclear Magnetic Imaging Study Stroke, March 1, 2002; 33(3): 825 - 830. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. A. Kent, V. M. Soukup, and R. H. Fabian Heterogeneity Affecting Outcome From Acute Stroke Therapy: Making Reperfusion Worse Stroke, October 1, 2001; 32(10): 2318 - 2327. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||