Homocysteine decreases blood flow to the brain due to vascular resistance in carotid artery

Abstract

An elevated level of Homocysteine (Hcy) is a risk factor for vascular dementia and stroke. Cysthathionine beta Synthase (CBS) gene is involved in the clearance of Hcy. Homozygous individuals for (CBS-/-) die early, but heterozygous for (CBS-/+) survive with high levels of Hcy. The gamma-Amino Butyric Acid (GABA) presents in the central nervous system (CNS) and functions as an inhibitory neurotransmitter. Hcy competes with GABA at the GABA(A) receptor and affects the CNS function. We hypothesize that Hcy causes a decrease in blood flow to the brain due to increase in vascular resistance (VR) because of arterial remodeling in the carotid artery (CA). Blood pressure and blood flow in CA of wild type (WT), CBS-/+, CBS-/+ GABA(A)-/- double knockout, and GABA(A)-/- were measured. CA was stained with trichrome, and the brain permeability was measured. Matrix Metalloproteinases (MMP-2 and MMP-9), tissue inhibitor of metalloproteinase (TIMP-3, TIMP-4), elastin, and collagen-III expression were measured by real-time polymerase chain reaction (RT-PCR). Results showed an increase in VR in CBS-/+/GABA(A)-/-double knockout>CBS-/+/>GABA(A)-/- compared to WT mice. Increased MMP-2, MMP-9, collagen-III and TIMP-3 mRNA levels were found in GABA(A)-/-, CBS-/+, CBS-/+/GABA(A) double knockout compared to WT. The levels of TIMP-4 and elastin were decreased, whereas the levels of MMP-2, MMP-9 and TIMP-3 increased, which indirectly reflected the arterial resistance. These results suggested that Hcy caused arterial remodeling in part, by increase in collagen/elastin ratio thereby increasing VR leading to the decrease in CA blood flow.

Figure 1

Genotype GABA_A-/+ and CBS-/- mice with specific sets of primers. Mr is 100 bp ladder. GABA has 280bp product. CBS -/+ has 1500bp and 800bp product.

Figure 2

Vascular flow and vascular resistance in carotid artery. (A) Flow was measured in WT, CBS-/+, CBS-/+ / GABA_A -/-, GABA_A -/- (A) by transonic probe snuggled around carotid artery. There was significant increase in VR in CBS-/+ and CBS-/+ / GABA_A -/- mice compared to WT. (B)The bar graph represents vascular resistance mean ± SEM in three experiments. *p<0.05 compared to WT.

Figure 3

(A) Histological analysis of carotid artery in wild type and CBS -/+ mice. Tissue section was labeled with trichrome for collagen. Note: the arrow indicates increased blue stain for collagen in CBS -/+ mice. Magnification x40. (B) Bar graph represents water content (%) in the brain cortex in WT, CBS-/+, CBS-/+ / GABA_A -/-, GABA_A -/- and CBS-/+ mice. Mean ± SEM (n=3). *p<0.05 represent compared to WT, # p<0.05 compared to CBS-/+ /GABA_A -/- double knockout mice.

Figure 4

Photograph taken on in vivo fluorescent microscope. Fluorescence signal shows that in HHcy (B) mice the blood flow deceases and increase in leakage for FITC in brain interstitial.

Figure 5

Represents Ct values for MMP-2 (A), MMP-9 (B), TIMP-3 (C) and TIMP-4(D) in WT, CBS-/+, CBS-/+ / GABA_A -/- and GABA_A -/-. Lesser the value of Ct represents more the quantity of gene. One Ct difference signifies two fold increases in gene quantity. There is significant increase in MMP-2, MMP-9 activity or decrease in Ct values. There is significant increase in TIMP-4 and decrease in TIMP-3 expression or decrease in Ct values for TIMP-4 and increase for TIMP-3. The bar graph represents mean ± SEM in three experiments. *p<0.05 compared to WT.

Figure 6

Represents Ct values for Collagen-III (A) and Elastin (B) in WT, CBS-/+, CBS-/+ / GABA_A -/- and GABA_A -/-. Lesser the value of Ct represents more the quantity of gene. One Ct difference signifies two fold increases in gene quantity. There is significant increase in collagen-III and decrease in elastin expression or decrease in Ct values for collagen-III and increase for elastin. The bar graph represents mean ± SEM in three experiments. *p<0.05 compared to WT.