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.

Ghrelin in central neurons.

Ghrelin, an orexigenic peptide synthesized by endocrine cells of the gastric mucosa, is released in the bloodstream in response to a negative energetic status. Since discovery, the hypothalamus was identified as the main source of ghrelin in the CNS, and effects of the peptide have been mainly observed in this area of the brain. In recent years, an increasing number of studies have reported ghrelin synthesis and effects in specific populations of neurons also outside the hypothalamus. Thus, ghrelin activity has been described in midbrain, hindbrain, hippocampus, and spinal cord. The spectrum of functions and biological effects produced by the peptide on central neurons is remarkably wide and complex. It ranges from modulation of membrane excitability, to control of neurotransmitter release, neuronal gene expression, and neuronal survival and proliferation. There is not at present a general consensus concerning the source of ghrelin acting on central neurons. Whereas it is widely accepted that the hypothalamus represents the most important endogenous source of the hormone in CNS, the existence of extra-hypothalamic ghrelin-synthesizing neurons is still controversial. In addition, circulating ghrelin can theoretically be another natural ligand for central ghrelin receptors. This paper gives an overview on the distribution of ghrelin and its receptor across the CNS and critically analyses the data available so far as regarding the effects of ghrelin on central neurotransmission.

Apoptosis in the mammalian CNS: Lessons from animal models.

It is generally assumed that about half of the neurons produced during neurogenesis die before completion of maturation of the central nervous system (CNS). Neural cell death is also relevant in aging and several neurodegenerative diseases. Among the modalities by which neurons die, apoptosis has very much attracted the interest of investigators because in this type of cell death neurons are actively responsible for their own demise by switching on a number of genes and activating a series of specific intracellular pathways. This review focuses on the cellular and molecular mechanisms of apoptosis in normal and transgenic animal models related to naturally occurring neuronal death within the CNS. We will also consider some examples of apoptotic cell death in canine neuropathologies. A thorough analysis of naturally occurring neuronal death in vivo will offer a basis for parallel and future studies involving secondary neuronal loss such as those in neurodegenerative disorders, trauma or ischaemia.

In vivo cellular and molecular mechanisms of neuronal apoptosis in the mammalian CNS.

Apoptosis has been recognized to be an essential process during neural development. It is generally assumed that about half of the neurons produced during neurogenesis die before completion of the central nervous system (CNS) maturation, and this process affects nearly all classes of neurons. In this review, we discuss the experimental data in vivo on naturally occurring neuronal death in normal, transgenic and mutant animals, with special attention to the cerebellum as a study model. The emerging picture is that of a dual wave of apoptotic cell death affecting central neurons at different stages of their life. The first wave consists of an early neuronal death of proliferating precursors and young postmitotic neuroblasts, and appears to be closely linked to cell cycle regulation. The second wave affects postmitotic neurons at later stages, and is much better understood in functional terms, mainly on the basis of the neurotrophic concept in its broader definition. The molecular machinery of late apoptotic death of postmitotic neurons more commonly follows the mitochondrial pathway of intracellular signal transduction, but the death receptor pathway may also be involved.Undoubtedly, analysis of naturally occurring neuronal death (NOND) in vivo will offer a basis for parallel and future studies aiming to elucidate the mechanisms of pathologic neuronal loss occurring as the result of conditions such as neurodegenerative disorders, trauma or ischemia.

Synapse-independent and synapse-dependent apoptosis of cerebellar granule cells in postnatal rabbits occur at two subsequent but partly overlapping developmental stages.

It has long been known that cells in the external granular layer die during postnatal development of the cerebellum. More recent findings indicate that at certain developmental stages, cell death occurs upon activation of an apoptotic program. We show that cerebellar granule cells in rabbits undergo programmed cell death at two different stages of maturation. At postnatal day 5 (P5), granule cell precursors and pre-migratory granule cells in the external granular layer incorporate the S-phase markers 5-bromo-2'-deoxyuridine and 5-iodo-2'-deoxyuridine with a pattern that is dependent upon the interval between the administration of the two tracers. Within 12-24 h after proliferation a significant number of labeled cells show typical ultrastructural alterations of apoptosis. DNA electrophoresis and cleavage of poly-ADP-ribose polymerase confirm the activation of the apoptotic machinery. After Southern blotting and immunodetection, incorporated 5-bromo-2'-deoxyuridine is present at the level of low size DNA oligomers as soon as 12 h after cell division. Therefore, this apoptotic phase is intrinsic to external granular layer neurons and independent of synaptic interactions with targets.Apoptotic cells, although fewer in number, are detected also in the internal granular layer and tend to increase from P5 to P10. It seems unlikely that these cells undergo DNA fragmentation in the external granular layer and subsequently migrate to their final destination, considering the data on cell cycle kinetics and the rapid tissue clearance by the glia. Parallel fiber-Purkinje spine synapses are already present in the molecular layer at P5. Therefore, the post-migratory granule cells likely undergo apoptosis as a failure to make proper synaptic contacts in the forming molecular layer. We conclude that the massive apoptosis of pre-migratory cells likely has a role in regulating the size of this rapidly expanding population of pre-mitotic neurons. The less tumultuous cell death of post-mitotic granule cells in the internal granular layer appears to be linked to the formation of the mature synaptic circuitry of the developing cerebellar cortex.

Localization, morphology and ultrastructure of taste buds in the domestic duck (Cairina moschata domestica L.) oral cavity.

The Authors report on localization of the taste buds in epithelium of the palate (70%), the floor of the oral cavity (28%) and the tongue (2%) in Domestic Duck. Each taste bud is oval-shaped, measuring roughly 130 x 60 microm, and communicates with the oral cavity by a short duct. There is topographical correspondence between the buds and the parietal salivary glands. Ultrastructural examination showed the following 4 cell types: 1) light cells--characterized by cytoplasm containing sparse perinuclear filaments, numerous light vesicles, a uniformly granular nucleus. 2) dark cells--with electron-dense cytoplasm, numerous perinuclear filaments, occasional light vesicles, an irregular and densely granular nucleus. 3) intermediate cells--with characteristics common to the two previous cell types. 4) basal cells--located at the ventral part of the button.

Apoptosis of undifferentiated progenitors and granule cell precursors in the postnatal human cerebellar cortex correlates with expression of BCL-2, ICE, and CPP32 proteins.

Naturally occurring apoptotic cells have been demonstrated in the postnatal cerebellum of rodents (Wood et al. [1993] Neuron 11:621-632; Krueger et al. [1995] J. Neurosci. 15:3366-3374). The nature of these cells differs among species: they are considered to be granule cells in mouse and astrocytes in rat. We labeled proliferating and apoptotic cells in the postnatal human cerebellar cortex by using antibodies against the Ki-67/proliferating cell nuclear antigen and the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling method for fragmented DNA. We also immunocytochemically detected some proteins encoded by genes modulating apoptosis and specific markers of neuronal/glial differentiation. Proliferating cells were observed from birth to 4 months, representing 31-35% of cells within the external granular layer (EGL). Apoptotic cells were detected during the first 3 months and corresponded to 5-7% of EGL cells. Much lower percentages were calculated in other cortical layers and white matter. The balance between proliferation and apoptosis was quantitatively favorable to the latter during the first postnatal week. Expression of BCL-2, CPP32, and interleukin-1beta-converting enzyme (ICE) proteins was spatially and developmentally regulated in parallel with apoptosis. Apoptotic cells were often CPP32/ICE immunoreactive but negative for BCL-2. Some apoptotic cells were positive for vimentin and, less frequently, for alpha-internexin or type-III beta tubulin, but never expressed the glial fibrillary acidic protein. This study demonstrates that apoptosis is a significant phenomenon in early postnatal development of human cerebellar cortex and shares some of the regulatory mechanisms described in other vertebrates.

The neurochemical maturation of the rabbit cerebellum.

The immunocytochemical distribution of several neuronal and glial antigens was investigated in the cerebellum of the developing and adult rabbit. Neurofilament positive neurons appeared at embryonic day (E) 25. Purkinje cells transiently expressed neurofilament polypeptides from postnatal day (P) 0 to 15. At later postnatal ages, staining was localised to the parallel fibres, the axonal arbors of the basket cells and fibres of the white matter. Neuron specific enolase (NSE) immunoreactivity was first detected at E25. At P0 Purkinje cells were positive and their staining intensity increased up to P25. From P30 to adulthood virtually all cells in the molecular and Purkinje cell layers were stained. Scattered PGP 9.5-immunoreactive neurons appeared in the cerebellar anlage at P25. Purkinje and Golgi cells were labelled by P0. Synaptophysin immunoreactivity was first observed at P0 in the form of a fine punctate reaction surrounding the perikarya and proximal dendrites of Purkinje cells. By P10, it became particularly intense within the cerebellar glomeruli of the granular layer. Neurons of the deep cerebellar nuclei expressed NSE and PGP 9.5 starting from E25. GFAP and S-100 immunoreactivities were first detected at P10. GFAP-immunopositive astrocytes progressively increased in number up to adulthood. S-100-immunoreactive glial cells were detected throughout the white and grey matter. Bergmann glial cells and their fibres were strongly immunoreactive. Vimentin positive glial cells and fibres were first observed at E15 and persisted up to adulthood. Double labelling experiments using a monoclonal antibody against the proliferating cell nuclear antigen (PCNA), a cyclin synthesised by mitotic cells, showed that neuronal and/or glial polypeptides are expressed only by fully differentiated postmitotic cells. These results indicate that major events in the neurochemical maturation of the rabbit cerebellum occur during the first month after birth, when the same pattern of the adult animal is attained.

Cell proliferation in the post-natal and adult mammalian central nervous system.

In the mammalian central nervous system cell proliferation is generally linked to developmental processes that are ultimated in the perinatal period. Few exceptions to this rule are known in certain regions of the mammalian brain, namely the post-natal cerebellar cortex and the adult subependymal layer. We report here the results of our studies about cell proliferation and related phenomena in these regions. Cell proliferation was visualised after bromodeoxyuridine incorporation and labeling of the proliferating cell nuclear antigen (PCNA), an endogenous protein expressed during the cell cycle. The occurrence of programmed cell death in the post-natal cerebellar cortex and the persistence of the embryonic isoform of neural cell adhesion molecule (NCAM) associated with proliferating cells in the adult subependymal layer were also investigated.

On the presence and ultrastructure of valves in the lymphatic vessels of the canine ovary.

The origin and course of the intraovarian lymphatic vessels were examined by means of histologic studies of the gonads of pubertal female dogs. The small vessels that were present in the cortex near the follicles and corpora lutea flowed into an anastomotic network located at the margins of the medulla. Vessels of larger caliber were found to originate from the medulla and to reach the hilum by an approximately rectilinear pathway. In concordance with their previous findings in other species, the authors identified numerous semilunar valves in the lymphatic vessels of the cortex. Their ultrastructure is described.