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1 Istituto di ricovero e cura a carattere scientifico Neuromed, Polo Molisano University La Sapienza, Pozzilli (Isernia)
2 Unità Operativa Cardiologia, IInd School of Medicine, University La Sapienza, Rome, Italy
3 Max Delbrück Center for Molecular Medicine, Berlin-Buch, Germany
4 Department of Neurological Sciences, Ist School of Medicine, University La Sapienza, Rome
5 Department of Pharmacological Sciences, School of Pharmacy, University of Milan, Milan
6 Department of Pathology, Ist School of Medicine, University La Sapienza, Rome, Italy
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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genetics; quantitative trait locus; functional genomics
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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We assessed the functional significance of the congenic segments by recording the stroke latency under a stroke-permissive dietary regimen (stroke proneness phenotype) as previously reported (12). The BP response to a high-salt diet was registered by a noninvasive procedure. We also measured protein excretion in the urine, since proteinuria usually precedes the onset of cerebrovascular events in the SHRsp strain (5, 21).
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METHODS |
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Congenic Lines
To construct our congenic lines, we selected both the whole region included between Klk and Mt1pa markers (42.3 cM) and the inner areas either between Klk and D1Mit3 (22 cM) or between D1Rat134 and Mt1pa (25 cM). As a result, all lines shared an overlapping area included between D1Rat134 and D1Mit3 (4 cM). We designed six congenic lines. Three lines carried the whole QTL, the Klk-D1Mit3 area, and the D1Rat 134-Mt1pa area, respectively, in the SHRsp configuration within the SHRsr background, and they were defined as follows: [SHRsr.SHRsp-(Klk-Mt1pa)], [SHRsr.SHRsp-(Klk-D1Mit3)], and [SHRsr.SHRsp-(D1Rat134-Mt1pa)]. The reciprocal lines carried the corresponding segments in the SHRsr configuration within the SHRsp background: [SHRsp.SHRsr-(Klk-Mt1pa)], [SHRsp.SHRsr-(Klk-D1Mit3)], and [SHRsp.SHRsr-(D1Rat134-Mt1pa)].
We generated the lines by crossing SHRsp with SHRsr in a reciprocal design to form heterozygous F1 progenies. The F1 progenies were subsequently backcrossed to either SHRsp or SHRsr strain (BC1). The genotype of the BC1 progenies was determined by genome screening with chromosome-specific informative microsatellite markers at 10-cM intervals. Heterozygotes at loci of interest (within the targeted area) were selected and backcrossed again to SHRsp or SHRsr (BC2). We repeated the backcrossing 10 times, always selecting heterozygous breeders for the next backcross while performing a genome scan on the other chromosomes. During this process, >99.9% of loci not undergoing selection became homozygous for one strain, while the selected allele from the other strain remained heterozygous. After BC10, two heterozygotes were crossbred, and the resulting offspring homozygous at loci of interest were bred to each other, thus fixing the alleles of interest in the homozygous state on the background of the other strain.
Genotyping
Genomic DNA was prepared from tail clipping of each animal by salt precipitation, as previously described (12). Microsatellite markers belonging to chromosome 1 (n = 21) and to the remaining chromosomes (n = 120) were genotyped in congenic animals to insure their inbreeding status outside of the selected congenic intervals. Genotyping was carried out by PCR amplification of 50 ng of DNA. The forward primer was labeled with [32P]ATP (DuPont, NEN) using T4 polynucleotide kinase (Promega). The PCR reactions were processed on a thermal cycler (model PTC 100; MJ Research, Watertown, MA), and the products were loaded onto a 7% polyacrylamide gel, run using a Base Ace apparatus (Stratagene, La Jolla, CA) at 60 W (Feathervolt 3000, Stratagene) for 4 h and exposed to XAR-6 film (Kodak, Rochester, NY) for autoradiography.
Phenotyping
At the age of 6 wk, the JD was initiated and maintained throughout the remainder of the experiment (3 mo) in all rat lines. The following animals were used: SHRsr, n = 18; SHRsp, n = 31; [SHRsr.SHRsp-(Klk-Mt1pa)], n = 33; [SHRsr.SHRsp-(Klk-D1Mit3)], n = 30; [SHRsr.SHRsp-(D1Rat134-Mt1pa)], n = 31; [SHRsp.SHRsr-(Klk-Mt1pa)], n = 33; [SHRsp.SHRsr-(Klk-D1Mit3)], n = 31; [SHRsp.SHRsr-(D1Rat134-Mt1pa)], n = 35.
The Japanese dietary regimen has been long used as the most suitable tool to induce stroke in the SHRsp strain (5, 10, 12, 21). The stroke phenotype was recorded, as previously described (12), as the latency to develop cerebrovascular events after the experimental protocol (stroke proneness) was initiated. Therefore, animals were monitored for signs of cerebrovascular events, as documented by presence of mono-, para-, hemi-, or quadriparesis, lethargy, akinesia, or sudden death. Moreover, animals were killed upon the appearance of symptoms of stroke. The whole brain was fixed in freshly prepared 4% paraformaldehyde. After fixation, each brain was sectioned in a coronal plane and embedded in paraffin. Histological sections (7-µm thick) were stained with hematoxylin-eosin and observed under light microscopy.
BP, Body Weight, and Proteinuria Measurements
BP was measured throughout the experiment in conscious, restrained rats at 2-wk intervals by tail-cuff plethysmography (BP 2000, Apex). Multiple BP measurements were made by the same operator at each time point, as a rule between 10:00 and 14:00, and the mean value was taken as the representative systolic BP level.
Urine was collected from rats of parental and congenic lines by housing them in individual cages at each time point. Protein concentration was measured according to Bradford (2) with bovine albumin as standard.
Finally, rats were weighed regularly throughout the experiment.
Statistical Analysis
BP, body weight (BW), and proteinuria data are provided as means ± SD. Between group analysis was performed by paired t-test or one-way analysis of variance, as applicable. Post hoc analysis was conducted using Scheffé’s multiple-comparison test. Survivor function was estimated by the life-table method. Log-rank and Wilcoxon statistics were used for testing equality of survivor functions. Statistical significance was stated at the P < 0.05 level. Analyses were carried out using Stata 8.2 (Texas Corp, 2005).
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RESULTS |
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BP, Proteinuria, and BW in Parental and Congenic Strains
BP tail-cuff measurements.
Systolic BP levels are shown in Table 1. They were similar at baseline (6 wk of age) in all lines. The BP increase under JD was similar in both parental and congenic lines.
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DISCUSSION |
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Few potential candidate genes are known to map within the chromosome 1 area isolated in the congenic lines. Among them, the genes encoding adrenomedullin and insulin-like growth factor II receptor deserve particular attention. In fact, they have been shown to be highly expressed in the injured brain following middle cerebral artery occlusion in the rat model (20, 22) and to have a neuroprotective effect in ischemic brain (9). Although structural differences of the adrenomedullin gene do not exist between the original parental strains (13), it remains to be determined whether its gene expression is under allele specific regulation.
One of the main purpose of the congenic strategy is to narrow down the chromosomal span of the original QTL interval. The region of interest is quite large, and the present definition of the lower border of the reciprocal congenic lines that are presented is incomplete. It well may be that the borders in the reciprocal congenic lines may differ outside the QTL region. Despite considerable efforts, so far, we have been unable to identify additional polymorphic markers mainly due to the high degree of genetic homogeneity between the two closely related rat parental strains. However, with increasing single nucleotide polymorphism markers being generated for the rat, this may become feasible in the future. Interestingly, even at the present stage, our findings suggest the relevance of a distinct chromosomal segment within the entire region that modulates stroke resistance and latency. A clear-cut stroke phenotype was related to the D1Mit3-Mt1pa area, which is specific only to the [SHRsp.SHRsr-(Klk-Mt1pa)] and [SHRsp.SHRsr-(D1Rat134-Mt1pa)] lines, showing a significant stroke resistance and, to their reciprocal strains, showing a significant stroke susceptibility. On the other hand, the observation that the [SHRsp.SHRsr-(Klk-D1Mit3)] line had a certain degree of stroke resistance whereas its reciprocal line did not show increased susceptibility to stroke raises interesting perspectives about the possible relevance of the D1Rat134-D1Mit3 area (which is shared by all lines).
In this regard, it is likely that increased susceptibility to stroke may require the interaction of multiple susceptibility variants in the target region of chromosome 1, and, therefore, replacement of just one of these variants with a single resistance variant may be sufficient to protect against stroke. Conversely, if interaction of multiple variants is necessary to confer susceptibility to stroke, transfer of only one of these variants from SHRsp onto the SHRsr background will not be sufficient to increase susceptibility to stroke. Thus it is possible that the D1Rat134-D1Mit3 region still contains an important variant even though an effect was observed in only one of the reciprocal congenics for that area.
The relevance of the findings obtained from the lines carrying the Klk-D1Mit3 segment may also underscore the importance of the genetic background as a critical factor influencing the phenotypic expression in congenic strains.
Overall, the data we provide make detailed genetic approaches aimed at the identification of the underlying gene(s) possible. This includes the development of congenic sublines for the chromosomal region of interest, as well as the identification of differentially expressed genes within the area of interest. In particular, the generation of nonoverlapping congenic substrains may help us to formally test the hypothesis that increased susceptibility to stroke requires the interaction of multiple susceptibility variants in the target region of chromosome 1.
This information may be important to the human disease. In fact, we have already demonstrated the close resemblance between the SHRsp and human stroke with regard to the gene encoding atrial natriuretic peptide (14, 15, 17).
The SHRsp strain shows high susceptibility to develop renal damage before the onset of cerebrovascular lesions (5, 18, 21). It has been suggested that renal lesions might be involved in the pathogenesis of stroke as a potential intermediate phenotype. According to the proteinuria levels detected in our experimental context, an association between the renal and the cerebrovascular phenotypes can be clearly confirmed. In fact, the SHRsp-derived lines had a delayed occurrence of proteinuria compared with the original strain, whereas two out the SHRsr-derived lines showed a rise of proteinuria only at the time of stroke events (which occurred 2 or 3 mo after starting JD). Thus our new models may provide an interesting tool to clarify the role of proteinuria in the pathogenesis of stroke. Moreover, it will be interesting to determine whether the same gene modulates both proteinuria and stroke occurrence in the SHRsp strain.
The issue of a stroke phenotype independence of BP, as previously reported in our experimental set-up (12), remains an interesting aspect to explore. We performed a noninvasive BP measurement throughout the experiment and obtained evidence in support of a lack of correlation between hypertension and stroke in our animal models. However, recently published American Heart Association recommendations caution against reliance on tail-cuff BP measurements in experimental animals to make claims about the BP independence of any phenotype (8). Thus we are aware that, in the absence of BP telemetry measurements, a clear definition regarding the relationship between stroke phenotype and BP under the effect of our stroke/QTL cannot be definitively assessed. By use of telemetry in future experiments aimed at the recording of all BP fluctuations in relation to stroke occurrence, our new animal models may provide important information relevant to this issue.
Finally, an interesting observation was obtained from monitoring the BW while the rats ingested the JD. We observed that the SHRsp-derived lines with a major stroke resistance also had increased BW and kept growing during JD compared with their strain of origin. On the other hand, the SHRsr-derived lines with a major stroke susceptibility had reduced growth compared with SHRsr. We suggest two possible explanations for these findings: there is an allelic influence on BW within the QTL on rat chromosome 1 or it is merely a secondary effect.
Earlier epidemiological studies suggested that coronary disease is a greater health-care burden than stroke. However, the recently reported Oxford Vascular Study demonstrated that this is not the case (11). The study showed high rates of acute vascular events outside the coronary arterial territory and a steep rise in event rates with age in all territories. As a matter of fact, stroke was no less common than coronary disease. The epidemiological findings have implications for prevention strategies, clinical trial design, and experimental research. Our experimental study opens new mechanistic perspectives. On the basis of our previous linkage data, we provided functional evidence for the role of a chromosome-1 QTL on stroke susceptibility. This result represents a promising background for the final identification of the specific components that directly contribute to stroke susceptibility in this rat model of a complex human disease. We are currently pursuing more detailed molecular genetic approaches to identify specific stroke-susceptibility and stroke-protection genes.
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GRANTS |
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FOOTNOTES |
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Addresses for reprint requests and other correspondence: N. Hubner, Max Delbrück Center for Molecular Medicine, 13092 Berlin, Germany (e-mail: nhuebner{at}mdc-berlin.de) and S. Rubattu, IRCCS Neuromed, 86077 Pozzilli (Is), Italy (e-mail: rubattu.speranza{at}neuromed.it).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* S. Rubattu and N. Hubner contributed equally to this work. 
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  M. Packard, Y. Saad, W. T. Gunning, S. Gupta, J. Shapiro, and M. R. Garrett Investigating the effect of genetic background on proteinuria and renal injury using two hypertensive strains Am J Physiol Renal Physiol, April 1, 2009; 296(4): F839 - F846. [Abstract] [Full Text] [PDF]
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 H. Yao, Z.-H. Cui, J. Masuda, and T. Nabika Congenic removal of a QTL for blood pressure attenuates infarct size produced by middle cerebral artery occlusion in hypertensive rats Physiol Genomics, June 19, 2007; 30(1): 69 - 73. [Abstract] [Full Text] [PDF]
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