Biochemical markers in vascular cognitive impairment associated with subcortical small vessel disease - A consensus report.

BACKGROUND: Vascular cognitive impairment (VCI) is a heterogeneous entity with multiple aetiologies, all linked to underlying vascular disease. Among these, VCI related to subcortical small vessel disease (SSVD) is emerging as a major homogeneous subtype. Its progressive course raises the need for biomarker identification and/or development for adequate therapeutic interventions to be tested. In order to shed light in the current status on biochemical markers for VCI-SSVD, experts in field reviewed the recent evidence and literature data. METHOD: The group conducted a comprehensive search on Medline, PubMed and Embase databases for studies published until 15.01.2017. The proposal on current status of biochemical markers in VCI-SSVD was reviewed by all co-authors and the draft was repeatedly circulated and discussed before it was finalized. RESULTS: This review identifies a large number of biochemical markers derived from CSF and blood. There is a considerable overlap of VCI-SSVD clinical symptoms with those of Alzheimer's disease (AD). Although most of the published studies are small and their findings remain to be replicated in larger cohorts, several biomarkers have shown promise in separating VCI-SSVD from AD. These promising biomarkers are closely linked to underlying SSVD pathophysiology, namely disruption of blood-CSF and blood-brain barriers (BCB-BBB) and breakdown of white matter myelinated fibres and extracellular matrix, as well as blood and brain inflammation. The leading biomarker candidates are: elevated CSF/blood albumin ratio, which reflects BCB/BBB disruption; altered CSF matrix metalloproteinases, reflecting extracellular matrix breakdown; CSF neurofilment as a marker of axonal damage, and possibly blood inflammatory cytokines and adhesion molecules. The suggested SSVD biomarker deviations contrasts the characteristic CSF profile in AD, i.e. depletion of amyloid beta peptide and increased phosphorylated and total tau. CONCLUSIONS: Combining SSVD and AD biomarkers may provide a powerful tool to identify with greater precision appropriate patients for clinical trials of more homogeneous dementia populations. Thereby, biomarkers might promote therapeutic progress not only in VCI-SSVD, but also in AD.

Clonal expansions of CD8(+) T cells dominate the T cell infiltrate in active multiple sclerosis lesions as shown by micromanipulation and single cell polymerase chain reaction.

Clonal composition and T cell receptor (TCR) repertoire of CD4(+) and CD8(+) T cells infiltrating actively demyelinating multiple sclerosis (MS) lesions were determined with unprecedented resolution at the level of single cells. Individual CD4(+) or CD8(+) T cells were isolated from frozen sections of lesional tissue by micromanipulation and subjected to single target amplification of TCR-beta gene rearrangements. This strategy allows the assignment of a TCR variable region (V region) sequence to the particular T cell from which it was amplified. Sequence analysis revealed that in both cases investigated, the majority of CD8(+) T cells belonged to few clones. One of these clones accounted for 35% of CD8(+) T cells in case 1. V region sequence comparison revealed signs of selection for common peptide specificities for some of the CD8(+) T cells in case 1. In both cases, the CD4(+) T cell population was more heterogeneous. Most CD4(+) and CD8(+) clones were represented in perivascular infiltrates as well as among parenchymal T cells. In case 2, two of the CD8(+) clones identified in brain tissue were also detected in peripheral blood. Investigation of the antigenic specificities of expanded clones may help to elucidate their functional properties.

Alterations in expression of glutamatergic transporters and receptors in sporadic Alzheimer's disease.

Excitatory neurotransmitter dysfunction has been discussed to be involved in the pathophysiology of Alzheimer's disease (AD). In the current study we investigated gene and protein expression patterns of glutamatergic receptors and transporters in brains of AD patients in various stages of disease using gene chip arrays, real time PCR and immunohistochemistry. We found marked impairment in the expression of excitatory amino acid transporters (EAAT1 and EAAT 2) at both gene and protein levels in hippocampus and gyrus frontalis medialis of AD patients, already in early clinical stages of disease. The loss of EAAT immunoreactivity was particularly obvious in the vicinity of amyloid plaques. In contrast, EAAT expression was up-regulated in the cerebellum of these patients. Furthermore, a significant up-regulation of the glutamatergic kainate (GRIK4) receptor observed by gene arrays was confirmed by quantitative RT-PCR in late stages in the hippocampus of AD patients. Moreover, there were down-regulations of other glutamatergic receptors such as NMDA (GRINL1A) and AMPA (GRIA4) receptors. Our data show marked changes in the functional elements of the glutamatergic synapses such as glutamatergic receptors and transporters and indicate impaired glutamate clearing rendering neurons susceptible to excess extracellular glutamate and support further the involvement of excitotoxic mechanisms in the pathogenesis of AD.

A new insight on Al-maltolate-treated aged rabbit as Alzheimer's animal model.

Lack of an adequate animal model for Alzheimer's disease (AD) has limited an understanding of the pathogenesis of the disease and the development of therapeutic agents targeting key pathophysiological processes. There are undoubtedly few satisfactory animal models for exploring therapies targeting at amyloid beta (Abeta) secretion, deposition, aggregation, and probably the inflammatory response. However, an understanding of the complex events--tau, Abeta, oxidative stress, redox active iron, etc.--involved in the neuronal cell loss is still unclear due to the lack of a suitable animal model system. The use of neurotoxic agents particularly aluminum-organic complexes, especially Al-maltolate, expands the scope of AD research by providing new animal models exhibiting neurodegenerative processes relevant to AD neuropathology. Examination of different species of aged animals including the rapidly advancing transgenic mouse models revealed very limited AD-like pathology. Most other animal models have single event expression such as extracellular Abeta deposition, intraneuronal neurofilamentous aggregation of proteins akin to neurofibrillary tangles, oxidative stress or apoptosis. To date, there are no paradigms of any animal in which all the features of AD were evident. However, the intravenous injection of Al-maltolate into aged New zealand white rabbits results in conditions which mimics a number of neuropathological, biochemical and behavioral changes observed in AD. Such neurodegenerative effects include the formation of intraneuronal neurofilamentous aggregates that are tau positive, immunopositivity of Abeta, presence of redox active iron, oxidative stress and apoptosis, adds credence to the value of this animal model system. The use of this animal model should not be confused with the ongoing controversy regarding the possible role of Al in the neuropathogenesis, a debate which by no means has been concluded. Above all this animal model involving neuropathology induced by Al-maltolate provides a new information in understanding the mechanism of neurodegeneration.

Role of metals in neuronal apoptosis: challenges associated with neurodegeneration.

Apoptosis is a tightly controlled process in which cell death is executed through activation of specific signalling pathways. Within cells, there are positive and negative regulatory pathways of apoptosis, hence it is targeted as 'Double-edged sword', the balance between these pathways dictates the cell fate. The past decade has seen intense focus on the mechanisms of apoptosis. Many important observations on the various signalling pathways mediating apoptotic cell death have been made and our understanding of the importance of apoptosis in both normal growth and development and patho-physiology has greatly increased. In addition, mechanisms of metal-induced toxicity continue to be of interest given the ubiquitous nature of these contaminants. The purpose of this review is to summarize our current understanding of the apoptotic pathways that are initiated by metals in Alzheimer's disease. Increased understanding of metal-induced (direct) and metal-amyloid-beta (indirect) linked neuronal cell death through the formation of reactive oxygen species (ROS) is critical to illuminate mechanisms of metal-induced cell death, as well as the potential role of metal speciation in neurodegeneration.

Hereditary Pick's disease with the G272V tau mutation shows predominant three-repeat tau pathology.

Frontotemporal dementia and parkinsonism linked to chromosome 17 have been associated with mutations in the microtubule associated protein tau (MAPT or tau) gene. This disorder is characterized by a large spectrum of neuronal and glial tau lesions in different brain regions. Pick bodies were found in a family with hereditary Pick's disease with the G272V mutation and in several families with other tau mutations in exons 9 and 11-13. The biochemical composition of Pick bodies varies between these mutations. Until recently, no detailed biochemical characterization of G272V brain material was done owing to unavailability of fresh frozen brain material. We now report a detailed study using the immunohistochemistry, western blots and electron microscopy of two brains with the G272V mutation that recently became available. Both brains showed severe neuronal loss in the temporal cortex, whereas in the frontal cortex the loss was less; and abundant Pick bodies in the dentate gyrus of the hippocampus, and caudate nucleus. The Pick bodies consisted exclusively of three-repeat (3R) isoforms, as was demonstrated by isoform-specific antibodies and supported by western blot analysis of sarkosyl-insoluble tau. These observations confirm that this family diagnosed with hereditary Pick disease meets all the criteria for this condition, including the presence of Pick bodies that are unphosphorylated at Ser262 and contain twisted filaments with long periodicity consisting only of 3R tau.

Pineal and cortical melatonin receptors MT1 and MT2 are decreased in Alzheimer's disease.

The pineal hormone melatonin is involved in physiological transduction of temporal information from the light dark cycle to circadian and seasonal behavioural rhythms, as well as possessing neuroprotective properties. Melatonin and its receptors MT1 and MT2, which belong to the family of G protein-coupled receptors, are impaired in Alzheimer's disease (AD) with severe consequences to neuropathology and clinical symptoms. The present data provides the first immunohistochemical evidence for the cellular localization of the both melatonin receptors in the human pineal gland and occipital cortex, and demonstrates their alterations in AD. We localized MT1 and MT2 in the pineal gland and occipital cortex of 7 elderly controls and 11 AD patients using immunohistochemistry with peroxidase-staining. In the pineal gland both MT1 and MT2 were localized to pinealocytes, whereas in the cortex both receptors were expressed in some pyramidal and non-pyramidal cells. In patients with AD, parallel to degenerative tissue changes, there was an overall decrease in the intensity of receptors in both brain regions. In line with our previous findings, melatonin receptor expression in AD is impaired in two additional brain areas, and may contribute to disease pathology.

Regulation of ovarian 17 beta-hydroxysteroid dehydrogenase activity by gonadotropin.

Serum testosterone levels are elevated prior to the lutropin surge, and decline abruptly following the release of endogenous lutropin. To investigate this phenomenon, the activity of 17 beta-hydroxysteroid dehydrogenase, the enzyme directly related to testosterone production from androstenedione, was measured. This was done in immature rats in which follicular maturation and ovulation were induced by pregnant mare serum gonadotropin administration. It appears that the effect of the gonadotropin on the enzyme activity is sharply divided into two phases that match with the follicular and the luteal phases. One day following gonadotropin administration, there was already a 7.67-fold increase in the original activity which further increased 48 h following hormone administration. At the peak of the lutropin surge, when follicular development is at its maximum, a 18.44-fold increase was measured. The activity fell abruptly 10 h following ovulation, at a time when fresh corpora lutea are already present in the ovary. It seems that the elevation of serum testosterone followed by its abrupt decline, is directly related to the increased and decreased ovarian 17 beta-hydroxy-steroid dehydrogenase activity. The possible importance of the observed changes to the mechanism of the onset of puberty are discussed.

Decreased activity of hippocampal neurons in Alzheimer's disease is not related to the presence of neurofibrillary tangles.

Numerous studies have established the key role of the Golgi apparatus (GA) in post-translational processing, transport and targeting of proteins destined for secretion, lysosomes and plasma membranes. Moreover, several studies performed in our laboratories have shown that the size of the immunocytochemically detected neuronal GA is a reliable index of neuronal activity in aging, Alzheimer's disease (AD) and amyotrophic lateral sclerosis. It has been suggested that in AD there is decreased neuronal activity, e.g. in terms of glucose metabolism and protein synthetic capability. To further explore the hypothesis of decreased neuronal activity in AD, in this study the size of the GA was measured in pyramidal neurons of the CA1 area of the hippocampus of non-demented controls and AD patients. The size of the GA was measured separately in neurons with and without neurofibrillary tangles (NFT). Moreover, in order to establish a correlation between the density of NFT and the size of the GA, the density of extraneuronal NFT was determined around each neuron and related to the size of its GA. The results, quantified by image analysis, indicate that there is a significant reduction in GA size in the neurons of the CA1 area of the hippocampus of AD patients. However, there was no significant relationship between the size of the GA and the presence or absence of intracellular NFT. In addition, there was no correlation between the density of extracellular NFT and GA size of adjacent neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Selective deposition of mutant tau in the FTDP-17 brain affected by the P301L mutation.

Frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) is a familial neurological disorder exhibiting autosomal dominant inheritance. Linkage analyses have led to the identification of many exonic and intronic mutations in the tau gene in affected families. Because FTDP- 17 causes extensive neuronal loss and intracellular tau deposits in affected regions, investigation of this disease should provide an important insight into the significance of tau deposits leading to neurodegeneration. Using site-specific antibodies that distinguish between wild-type and mutant tau, we have analyzed the proportions of wild-type and mutant tau in the soluble and insoluble fractions of the P301L brain. Western blotting showed that mutant tau was selectively deposited in the Sarkosyl-insoluble fraction. Consistent with this, immunocytochemistry showed that intraneuronal tau deposits consisted exclusively of mutant tau. In one case in which abundant senile plaques occurred, in addition to mutant tau, small amounts of wild-type tau were also deposited. On the other hand, the protein levels of mutant tau in the soluble fraction were selectively decreased despite no detectable decrease in the levels of mutant tau mRNA.

ApoE epsilon4 genotype is accompanied by lower metabolic activity in nucleus basalis of Meynert neurons in Alzheimer patients and controls as indicated by the size of the Golgi apparatus.

We previously found apolipoprotein (apoE) epsilon4-dependent lower metabolic activity in nucleus basalis of Meynert (NBM) neurons in Alzheimer disease (AD). In the present study we examined the metabolic activity in the NBM of 39 mentally intact control subjects with different APOE genotype. The control subjects had either no AD pathology (Braak stage 0) or the very beginning of AD pathology (Braak stage I-II). We used the Golgi apparatus (GA) size as a measure of neuronal metabolic activity. Control subjects carrying an apoE epsilon4 allele showed reduced neuronal metabolism; they had significantly more neurons with smaller GA sizes compared to control subjects not carrying an apoE epsilon4 allele. Only control subjects not carrying an apoE epsilon4 allele had increased neuronal metabolism in Braak I-II subjects. They had more neurons with larger GA sizes compared to Braak 0 subjects, which may reflect a compensatory mechanism. Our data indicate that APOE epsilon4 may act by a lower neuronal metabolism as a risk factor for cognitive impairment in normal aging and early prodromal AD. As the disease progresses into later stages of AD (Braak V-VI) neuronal metabolism strongly diminishes, resulting in neurons with extremely small GA sizes, irrespective of APOE genotype.

Effect of androstanediol sulfates on luteinizing hormone release in ovariectomized rats.

Androstanediols are the major products of the immature rat ovary and are present in peripheral circulation mainly as sulfate conjugates. In this paper we identified 5 alpha-androstane-3 beta, 17 beta-diol-3-monosulfate (3 beta-A-MS) as one of the forms found in blood and subsequently synthesized and administered it to ovariectomized rats at a dose of 100 microgram/100 g BW . day from 21-45 days of age. This dose effectively inhibits postcastrational LH elevation. Other androstanediols examined, like 5 alpha-A-3 alpha, 17 beta-diol-disulfate, 5 alpha-A-3 beta, 17 beta-diol-disulfate, and the free 5 alpha-A-3 beta, 17 beta-diol do not exert such an effect on LH release. The MCR of 3 beta-A-MS was 441 +/- 64 ml/h, independent of the infusion rate between 0.15-15.0 microgram/h, and its production rate was calculated to be 37 microgram/day at the age of 30 days. The quantitative relations of the steroid level in serum to its capacity to inhibit LH release was studied using Silastic capsules. A steady concentration of 1.1 ng 3 beta-A-MS/ml serum inhibits postcastrational LH release in the immature female rat. Since a similar or higher concentration of the steroid is present in the intact rat, it is assumed that 3 beta-A-MS controls pituitary LH release in the intact immature female rat. (Endocrinology 108: 500, 1981)

Decreased number of vasopressin immunoreactive neurons in the medial amygdala and locus coeruleus of the aged rat.

The earlier described age-related decreases in vasopressin innervation of extra-hypothalamic rat brain regions were found to coincide with a decrease in vasopressin expression in the cells where these fibers originated. A significant age-related decrease in the number of vasopressin-immunoreactive cell bodies was found in the medial amygdala and locus coeruleus of senescent Brown-Norway (BN/BiRij) rats (33 months) when compared to middle-aged (19 months) and young (3 months) rats. In addition, total testosterone plasma levels were significantly reduced in middle-aged and old rats as compared to young animals and the number of vasopressin-immunoreactive cells in both the medial amygdala and locus coeruleus correlated significantly with the decreased testosterone levels in a similar way as found earlier for vasopressin terminals.

Increased levels of DNA breaks in cerebral cortex of Alzheimer's disease patients.

It has been hypothesized that Alzheimer's disease (AD) is caused by an accumulation of damage in DNA due to defective DNA-repair (21). Attempts to test this hypothesis by determining the activity of DNA-repair systems in nonneuronal cells from AD patients and controls so far provided conflicting results. An alternative approach is the direct comparison of DNA-damage levels in neuronal tissue of AD patients and controls. In the present study we assayed the level of DNA breaks and alkali-labile sites in cerebral cortex tissue samples from AD patients and controls obtained from rapid autopsies. Our data on 11 AD patients and 8 control subjects indicate an at least two-fold higher level of DNA damage in cortex of AD patients as compared to controls.

8OHdG levels in brain do not indicate oxidative DNA damage in Alzheimer's disease.

Accumulation of oxidative DNA damage has been proposed to underlie aging and neurodegenerative diseases such as Alzheimer's Disease (AD). The DNA adduct 8-hydroxy-2'-deoxyguanosine (8OHdG) is considered a good indicator of oxidative DNA damage. To investigate whether this type of DNA damage is involved in AD etiology, 8OHdG levels were determined in postmortem human brain tissue of controls and AD patients (in frontal, occipital, and temporal cortex and in hippocampal tissue). Parametric studies in rat revealed no influences of postmortem delay, repeated freezing/thawing or storage time. In human brain, approximately two 8OHdG molecules were present per 10(5) 2'-deoxyguanosines. In AD patients and controls, 8OHdG-levels were not related to age, sex, or brain region. Also, no differences were found between controls and AD patients. It was concluded that 8OHdG in nuclear DNA, although present throughout the brain in fairly high amounts, does not accumulate with age, nor does it appear to be involved in AD. More detailed studies are required to extend this conclusion to other types of oxidative damage.

Pathologic findings in a case of primary progressive aphasia.

We report the histopathologic and immunohistochemical findings of a patient who suffered from primary progressive aphasia for 13 years. During the course of his illness, he was diagnosed as having Pick's disease, based on gradually progressive mild personality changes and MRI findings of severe bilateral temporal lobe atrophy. There was severe neuronal loss in the temporal gyri, intense gliosis throughout the cortex, and mild gliosis of the temporal white matter, without any changes typical for Alzheimer's disease or Pick's disease. Using the antibody Alz-50, we found many Alz-50-positive neurons that exhibited a granular or diffuse cytoplasmic stain without fibrous structures in the temporal and parietal cortex, but no ubiquitin or beta (A4) protein-reactive cells, nor spongiform changes. Staining for Alz-50 and ubiquitin did not reveal the presence of Pick bodies or Lewy bodies. We consider this case to be an example of nonspecific cortical degeneration. Our findings stress the need for histopathologic verification of the primary progressive aphasia syndrome.

Histopathologic correlates of white matter changes on MRI in Alzheimer's disease and normal aging.

We investigated the histopathologic correlates of white matter changes in Alzheimer's disease (AD) patients (n = 6) and controls (n = 9) using postmortem MRI. White matter changes were rated on a 0 to 3 scale in 51 regions. Histopathologically, we subjectively rated the loss of myelinated axons in the deep and periventricular white matter, denudation of the ventricular ependyma, gliosis, width of the perivascular spaces, and leptomeningeal congophilic angiopathy; we measured structural changes in the walls of the blood vessels in the white matter in micrometers. The AD brains displayed significantly more white matter hyperintensities on MRI than controls. Histopathologically, the denudation of the ventricular ependyma and the gliosis were significantly more severe in AD than in controls, and there was a trend toward more loss of myelinated axons in the deep white matter in the AD brains (p = 0.07). The MRI abnormalities correlated with the loss of myelinated axons in the deep white matter (r' = 0.37; p < 0.01) and with the denudation of the ventricular lining (r' = 0.54; p < 0.01). We could not find any evidence for arteriolosclerosis, but the mean thickness of the adventitia of the arteries of the deep white matter in AD almost doubled the value in control brains (p = 0.0009). We conclude that white matter abnormalities in AD patients and controls consist of loss of myelinated axons, probably caused by arterial changes and breakdown of the ventricular lining. Since imaging/histopathologic correlation was similar in AD patients and controls, these changes probably represent some form of accelerated aging.

Histopathologic correlate of hypointense lesions on T1-weighted spin-echo MRI in multiple sclerosis.

Postmortem unfixed whole brains from five multiple sclerosis (MS) patients were examined by MRI using a T2- and T1-weighted spin-echo (SE) sequence and histology to investigate the histopathologic characteristics of hypointense lesions on T1-weighted SE MR images. The degree of hypointensity was scored semiquantitatively by two blinded observers in reference to normal-appearing white matter. Signal intensities of the lesions and the normal-appearing white matter were measured to obtain contrast ratios. Hematoxylin-eosin stain was used to assess degree of matrix destruction (decrease of density of the neuropil) and cellularity of a lesion, Klüver-Barrera stain for degree of demyelination, Bodian stain for axonal density, and immunostaining of glial fibrillary acid protein for reactive astrocytes and fibrillary gliosis. Nineteen lesions were selected for analysis. Nearly all lesions were compatible with the chronic MS plaque: hypocellularity, absence of myelinated axons, in the presence of reactive astrocytes. Contrast ratios of the lesions were highly correlated (R = -0.90; p < 0.01), with degree of hypointensity scored semiquantitatively. Degree of hypointensity on T1-weighted SE images did not correlate with degree of demyelination or number of reactive astrocytes, but was associated with axonal density (R = -0.71; p = 0.001). A trend was found with degree of matrix destruction (R = 0.45; p = 0.052). We conclude that, in our limited sample, hypointense lesions seen on T1-weighted SE MR images are associated histopathologically with severe tissue destruction, including axonal loss. Our results need to be substantiated in a larger study on more varied patient material to evaluate the use of hypointense lesions as a surrogate marker of persistent deficit in MS patients.

Familial aggregation in frontotemporal dementia.

OBJECTIVE AND BACKGROUND: Frontotemporal dementia (FTD) is a common, non-Alzheimer's dementia. Its familial occurrence has been reported, but the frequency of positive family history is unknown. METHODS: We carried out a nationwide genetic-epidemiologic study of FTD in the Dutch population of 15 million people. The family history of dementia was analyzed in 74 FTD patients and 561 age- and gender-matched control subjects. RESULTS: We found one or more first-degree relatives with dementia before age 80 in 38% (28 of 74) of FTD patients, but only in 15% (84 of 561) of control subjects. Ten percent of FTD patients had two or more first-degree relatives with dementia compared with 0.9% of the control subjects. Seven percent of FTD patients showed the ApoE4E4 genotype versus 2.3% of the control subjects. The first-degree relatives of FTD had a risk of 22% for dementia before age 80 compared with 11% in relatives of control subjects. The age of onset of dementia in affected first-degree relatives of FTD patients (60.9+/-10.6 years) was significantly lower than among affected relatives of control subjects (72.3+/-8.5 years). The first-degree relatives of FTD patients were 3.5 times (95% CI, 2.4 to 5.2) more at risk for developing dementia before age 80 than relatives of control subjects. The hazard ratio in the subgroup with unknown linkage to chromosome 17 was 2.4 (95% CI, 1.5 to 3.7). CONCLUSION: This study documents the importance of genetic factors in a proportion of FTD patients with the age at onset of dementia in first-degree relatives being 11 years earlier than in the general population.

Increased activity of hypothalamic corticotropin-releasing hormone neurons in multiple sclerosis.

Clinical observations and animal studies suggest that the hypothalamo-pituitary-adrenal (HPA) axis plays a role in the susceptibility to and the recovery from multiple sclerosis (MS). Since the HPA-axis is under the control of corticotropin-releasing hormone (CRH) neurons of the hypothalamus, we determined 2 parameters for activation of the CRH neurons in the hypothalamic paraventricular nucleus (PVN) in MS patients. Since the HPA-axis is more activated in MS, we expected an increased activity of CRH neurons. We also expected to see an age-related increase in CRH activity, because of the possible role of the HPA-axis in the age-related decrease in susceptibility to MS. The number of CRH cell profiles and the proportion of CRH neurons co-expressing vasopressin were used as parameters for activity. CRH cell population became more activated both in control and MS patients, from 40 years of age onwards, when the prevalence of MS starts to decrease in the population. The CRH neurons showed a significantly higher level of activation in MS patients than in controls, as appeared from the 3-fold increase in CRH cell number and the 4.5-fold increase in cells co-expressing CRH and vasopressin (AVP).