Serum Hcy was detected at levels within the range of reference human values in 45 aged rhesus monkeys, and was related to altered volumetric and microstructural tissue density indices in prefrontal, parietal, temporal, and subcortical areas previously found to be sensitive to presumed cerebrovascular changes. Hcy was non-significantly higher in our CR animals. A similar CR manipulation lasting 3 months also found a non-significant 6.43% increase in Hcy (Chen et al., 2009
Despite statistically comparable Hcy concentrations in the CR and control groups, the association between higher Hcy levels and lower global gray matter volume in the control group was not observed in CR animals. These results suggest that CR may potect the brain against the adverse affects of Hcy, or from effects of aging for which Hcy is a biomarker. Because CR downregulates arterial proteoglycans and shrinks low density lipoprotein size in this cohort of monkeys (Edwards et al., 1998
), these animals may have been protected against damage to the vascular endothelium and consequent regional neuronal ischemia that could affect brain tissue volumes. For example, CR may have an effect on basal nitric oxide levels because it is associated with endothelial health. Mice on a 35% CR diet following hindlimb ischemic surgery show increased nitric oxide synthase phosphorylation of endothelial cells (Kondo et al., 2009
), which could counteract Hcy induced decreases in nitric oxide that render microvascular lumen less distensible. Alternatively, CR could cause Hcy to be more efficiently converted into cysteine through either: 1) increased expression of the catalyzing agents Cystathionine β-synthase or Cystathionine β-lyase; or 2) higher concentrations of pyroxidine (vitamin B6
). Twenty-four hour fasting among rats raised cysteine levels compared to ad libitum
feeding in the same subjects, although the effect was non-significant (Smolin and Benevenga, 1984
With respect to regional gray matter volume, higher Hcy levels were related to a global maximum in a bilateral cluster encompassing anterior cingulate cortex. In the Framingham Offspring study, total Hcy levels were inversely related to frontal lobe volume (Seshadri et al., 2008
), and a voxel-based morphometry assessment of B vitamin supplementation specifically implicated a similar location in humans (Erickson et al., 2008
). Most additional anatomic regions identified in the latter study corresponded to our regional gray matter findings in monkeys. These clusters included parietal cortex and intraparietal areas, cingulate cortex, supplementary motor area, and the cingulate sulcus. Furthermore, an ROI analysis associated higher Hcy with lower right hippocampus volume in alcoholic patients (Wilhelm et al., 2008
). Ganguly and colleagues (2008)
also found that microdialysis infusion of Hcy in anaesthetized rats exacerbated post-stroke damage to the striatum. Peripheral Hcy was related to less gray matter volume in both of these structures in our sample.
For our diffusion tensor imaging measures, the relationship between Hcy and gray matter tissue density as estimated by mean diffusivity was similar to the volumetric results in dorsal premotor and anterior cingulate cortices, as well as in the orbital prefrontal cortex and paralimbic structures, most of which can be affected by age or non-age related pathologies that raise Hcy (Thomas et al., 2002; Sowell et al., 2003
; Shirpoor et al., 2009
). It is possible that some cerebrovascular pathology may directly contribute to the structural changes in some of these regions. In addition, several rodent models show that application of Hcy causes direct neurotoxic effects on cerebellar purkinje and granule cells in vitro
(Kim and Pae, 1996
; Oldreive and Doherty, 2007
Higher Hcy levels were associated with lower white matter volume and reduced fractional anisotropy in regions of efferent and afferent ponto-cerebellar connections and basis pontis. Higher radial diffusivity in the middle cerebellar peduncle and almost all other regions of altered fractional anisotropy suggested that these changes were more likely related to less myelin rather than fewer axons (Song et al., 2002
). Hcy-related changes in white matter volume, unlike gray matter, were not modified by CR, although focal protection was evident in cortical and sub-cortical areas. A few neurological syndromes demonstrate selective vulnerability of the pons, cerebellar peduncles, and splenium to cerebrovascular injury. The pons and posterior white matter are specifically prone to damage during osmotic stress or hypertensive crisis (Pearce, 2009
). Age-related cerebrovascular changes in the central pons (“pontine rarefaction”) has been associated with symptoms of disequilibrium in elders and attributed to the effects of cerebrovascular disease on penetrating pontine arterioles (Pullicino et al., 1995
; Kwa et al., 1998
). Hcy also appears to be a useful biomarker for periventricular and deep white matter hyperintensities in normal aging, as well as in neurodegenerative disorders (Sachdev, 2004
The pons and cerebellum have not typically been examined in these previous reports, but both were related to Hcy in our analyses. B12
deficiency, which results in elevated serum Hcy levels, can cause selective demyelination of the posterior columns of spinal cord, with similar abnormalities in the pons and cerebellar peduncles (Katsaros eta al., 1998
). More diffuse leukoencephalopathy, also with occipital/parietal predominance, has been described in a case of uncontrolled homocystinuria (Vatanavicharn et al., 2008
). Folate depletion versus repletion among murine dams raised Hcy levels and induced substantial cell reductions in pons and cerebellum of offspring (Xiao et al., 2005
). Higher levels of Hcy may be implicated in degrading coursing fibers of the cerebellothalamocortical or corticopontocerebellar tracts. Indeed, the premotor cortices, the body of the corpus callosum, mid cingulate, and thalamic bundles all showed lower fractional anisotropy as Hcy concentrations increased. Patients with multiple sclerosis manifest lower fractional anisotropy and higher mean diffusivity in the body of the corpus callosum, displaying dysregulation of activity in the above regions that grade for motor output (Lenzi et al., 2007
). More broadly, lower fractional anisotropy in the prefrontal cortices, the genu of the corpus callosum, and other regions appear to be associated with a variety of behavioral deficits including reduced motor speed, executive functioning, and working memory (Kennedy and Raz, 2009
Although hyperhomocysteinemia may directly induce the above effects, Hcy could alternatively serve as a risk marker rather than a true causal factor in its relationship to neural atrophy. Hcy can cause excitotoxicity by inducing oxidative stress, but it also sensitizes neurons to exogenous or endogenous toxins such as amyloid beta accumulation (Doherty, 2007
). Higher Hcy also potentiates proinflammatory cytokine expression, which can dysregulate intracellular energy homeostasis (Karalis et al., 2009
) and cause oxidative stress in a paracrine manner (Gokkusu et al., 2010). CR has been shown to reduce levels of these proteins in this monkey cohort (Willette et al., 2010) and non-obese adults on 20% CR diets (Fontana et al., 2007). While interleukin-6 levels did not mediate Hcy-related associations with neural regions in this sample (data not shown), CR may affect other inflammatory agents or similar factors that are potentiated by Hcy.
There are several limitations to the current study. Our MRI and physiological data were cross-sectional in nature and do not allow causal inferences to be made. Although we removed diffusion tensor imaging scans with artifacts in order to minimize the possibility of spurious results in voxel-wise analyses, this step could have inadvertently introduced selection bias in the interaction analyses for fractional anisotropy and mean diffusivity. We also did not collect gait or balance motor measures, which would have been a useful to regress onto the white matter volume map overlaying the pons and anterior cerebellum; future studies should examine this relationship.
In conclusion, this cross-sectional regression analysis of the associations among Hcy levels, gray matter and white matter volume, and microstructural density of parenchyma in rhesus monkeys confirms and extends previous ROI and lobar volume studies in humans. CR may provide protection from Hcy-related alterations in gray matter within prefrontal, parietal, and cerebellar regions. In contrast, CR did not appear to similarly lessen the impact of Hcy on white matter volume, although it did influence microstructural density of pontocerebellar fibers, corpus callosum, and other important tracts. CR may mitigate the Hcy-related volume loss in brain regions implicated in the development of movement, gait, and somatosensory difficulties with aging.