Neuropathology and Applied Neurobiology

Experimental bilateral carotid artery occlusion: a study of the optic nerve in the rat

The presence of marked changes in the neural retina and retinal vessels of Long‐Evans pigmented rat following bilateral carotid ligation has been previously documented by serial ophthalmologic examinations. Light and electron microscopic studies of the optic nerve in this experimental animal model revealed advanced optic nerve atrophy in ten of twelve rats examined. There was no significant pathology in either the retina or optic nerve in the remaining two rats after carotid ligation or in the sham‐operated controls.

Defects of mitochondrial function have been proposed as a potential mechanism in the development and pathogenesis of Alzheimer's disease (AD) and neuronal apoptosis. Mitochondrial enzyme‐deficient pyramidal neurones are found in greater quantities in the hippocampus of AD patients than in age‐matched controls. The presence of these neurones indicates that high levels of mutant mtDNA (mitochondrial DNA), sufficient to cause a biochemical deficiency within individual neurones, occur more frequently in AD than in normal ageing. This study analyses the relationship of cytochrome c oxidase (COX)‐deficient neurones with the neuropathological markers of AD, neurofibrillary tangles (NFTs) and amyloid plaques, as well as markers of neuronal apoptosis known to occur in AD brains. Frozen sections of hippocampi from three AD patients were used to directly colocalize in situ the presence of histochemically COX‐deficient neurones with immunohistology for the classical neuropathological markers of AD, tau and β‐amyloid. In addition, we also directly colocalized these mitochondrial‐enzyme deficient neurones using terminal deoxynucleotidyl transferase‐mediated dUTP nick end labelling and cleaved caspase‐3. The distribution of amyloid plaques is anatomically distinct from the COX‐deficient hippocampal pyramidal neurones and the neurones that contained NFTs or apoptotic labelling were always COX‐positive. COX‐deficient, succinate dehydrogenase‐positive hippocampal neurones indicative of high mtDNA mutation load do not appear to be prone to apoptosis or to directly participate in the over production of tau or β‐amyloid. Biochemically significant mitochondrial defects do occur in AD and are likely to contribute to the overall central nervous system dysfunction in impairing neuronal function and possibly causing neurodegeneration via mechanisms other than apoptosis.

Gliomas are the most frequent intrinsic tumours of the central nervous system and encompass two principle subgroups: diffuse gliomas and gliomas showing a more circumscribed growth pattern (‘nondiffuse gliomas’). In the revised fourth edition of the WHO Classification of CNS tumours published in 2016, classification of especially diffuse gliomas has fundamentally changed: for the first time, a large subset of these tumours is now defined based on presence/absence of IDH mutation and 1p/19q codeletion. Following this approach, the diagnosis of (anaplastic) oligoastrocytoma can be expected to largely disappear. Furthermore, in the WHO 2016 Classification gliomatosis cerebri is not an entity anymore but is now considered as a growth pattern. The most important changes in the very diverse group of ‘nondiffuse’ gliomas and neuronal‐glial tumours are the introduction of anaplastic pleomorphic xanthoastrocytoma, of diffuse leptomeningeal glioneuronal tumour and of RELA fusion‐positive ependymoma as entities. In the last part of this review, after very briefly touching upon classification of neuronal, choroid plexus and pineal region tumours, some practical implications and challenges associated with the WHO 2016 Classification of gliomas are discussed.

The development of the human blood‐brain and blood‐CSF barriers

The commonly held belief that the fetal blood‐brain and blood‐CSF barriers are immature is reviewed. Results obtained from carefully conducted experiments with horseradish peroxidase and optimal freeze‐fracturing suggest that the chick, rat and monkey brain barrier systems to proteins are tight from the earliest stages of development. Previous studies are reviewed in the light of new information on retrograde axonal transport, circumventricular organs, the proper use of horseradish peroxidase, freeze‐fracturing, immunocytochemistry and plasma protein gene expression in the developing human brain. Original data on the development of human brain barrier systems are included. Tight junctions between cerebral endothelial and choroid plexus epithelial cells form the morphological basis for these systems. CSF in the fetus contains a remarkably high concentration of protein in contrast to adult CSF which is characterized by a very low protein concentration. This has previously been interpreted as due to immaturity of barriers in the fetal brain. Tight junctions between cerebral endothelial cells and between choroid plexus epithelial cells have been investigated in human embryos and fetuses by freeze fracture and thin section electron microscopy. As soon as the choroid plexus and the brain capillaries differentiated they exhibited well formed tight junctions. These junctions were very complex at early stages of development. A new barrier consisting of ‘strap junctions’ was found in the developing germinal matrix. The very high concentration of protein in early human fetal CSF cannot be accounted for by a lack of tight junctions

During Wallerian degeneration, the Schwann cell basal laminal ensheathment around myelinated nerve fibres remains after the removal of myelin and axonal debris, forming a corrugated tube within which Schwann cell proliferation takes place. In nerve biopsies from patients with diabetic neuropathy, such residual basal laminal tubes tend to be circular rather than corrugated and appear to be more persistent during regeneration; this suggests increased rigidity and durability. These changes could be the result of increased cross–linkage of type IV collagen or alterations to other components of the basal lamina. A similar mechanism may be responsible for the thickening of perineurial basal laminae and the reduplication of basal laminae around endoneurial capillaries in diabetic patients; such reduplication may lead to reduced compliance of the vessel walls and impaired vascular perfusion.

The immunological basis of multiple sclerosis (MS) is well recognized but the factors inducing MS lesions are unclear. In this study, we test the hypothesis that focal brain injury, inflicted during the pre‐clinical stages of experimental allergic encephalomyelitis (EAE), will enhance the severity of immunological damage in the cerebral hemispheres and spinal cord. Acute EAE was induced in 30 Lewis rats by the injection of guinea pig spinal cord homogenate in complete Freund's adjuvant. A cryolesion to the surface of the left cerebral hemisphere was induced at 3 days (n=6) or 8 days (n=10) post‐inoculation (pi) and animals were killed at 15 days pi. Control animals were EAE only (n = 9), cryolesion only (n=4), EAE and sham cryolesion (n=5) and normal animals (n=3). Brain and spinal cord were stained by immunocytochemistry using W3/13 (T‐lymphocytes) OX6 (MHC Class II) and GFAP (astrocytes) antibodies. The results showed a 2‐fold increase in the number of EAE lesions in the brain with significant and widespread increase of MHC Class II antigen expression by microglia, in the cryolesion EAE 8 days p.i when compared with EAE only animals. The pattern of enhancement suggests that it is due to (i) local spread of tissue or serum factors from the cryolesion; (ii) neural factors affecting remote regions of the CNS; (iii) stimulation of the immune system which may occur due to products of brain injury draining to regional cervical lymph nodes. Investigation of the mechanisms involved may prove fruitful in establishing factors which initiate, aggravate or ameliorate brain damage in multiple sclerosis.

Multiple sclerosis and experimental autoimmune encephalomyelitis (EAE) are autoimmune inflammatory diseases in which cytokines are intimately involved. Here we test the hypothesis that injection of pro‐inflammatory cytokines, tumour necrosis factor‐α (TNFα) and interferon gamma (IFNγ) into the brain of animals in the prodromal phase of EAE significantly enhances inflammation in the central nervous system (CNS). We were particularly interested to learn whether a local increase in cytokines influenced the pathology locally, or more extensively, within the CNS. EAE was induced in female adult Lewis rats. Eight days post‐inoculation, TNFα or INFγ was injected into one cerebral hemisphere. Days 11 and 13 post‐inoculation (3 and 5 days after the injection of cytokine) inflammation was quantified by the number of perivascular cuffs and the degree of major histocompatibility complex (MHC) class II expression by microglia. Normal animals injected with cytokines, and EAE animals with saline injection served as controls. Results: microglial activation was increased three‐ to fourfold in the brain and eightfold in the spinal cord (P ≤ 0.05); lymphocyte invasion was increased sixfold in the brain and three‐ to fourfold in the spinal cord (P ≤ 0.01) following injections of TNFα or INFγ in EAE animals compared with controls. Significant axonal damage was observed in white matter associated with the perivascular cuffs. Conclusion: local changes in the release of pro‐inflammatory cytokines within the brain in EAE results in the widespread enhancement of autoimmune inflammation within the brain and cord, and exacerbation of clinical symptoms.

S. Nag, A. Kapadia and D. J. Stewart (2011) Neuropathology and Applied Neurobiology37, 3–23
Molecular pathogenesis of blood–brain barrier breakdown in acute brain injury

Historically, the blood–brain barrier (BBB) was considered to be at the level of cerebral endothelium. Currently, the interaction of endothelium with other components of the vessel wall and with neurones and glial cells is considered to constitute a functional unit, termed the neurovascular unit that maintains cerebral homeostasis in steady states and brain injury. The emphasis of this review is on cerebral endothelium, the best‐studied component of the neurovascular unit, and its permeability mechanisms in health and acute brain injury. Major advances have been made in unravelling the molecular structure of caveolae and tight junctions, both of which are components of the structural barrier to the entry of plasma proteins into brain. Time course studies suggest that caveolar changes precede junctional changes in acute brain injury. Additional factors modulating BBB permeability in acute brain injury are matrix metalloproteinases‐2 and 9 and angiogenic factors, the most notable being vascular endothelial growth factor‐A and angiopoietins (Ang) 1 and 2. Vascular endothelial growth factor‐A and Ang2 have emerged as potent inducers of BBB breakdown while Ang1 is a potent anti‐leakage factor. These factors have the potential to modulate permeability in acute brain injury and this is an area of ongoing research. Overall, a combination of haemodynamic, structural and molecular alterations affecting brain endothelium results in BBB breakdown in acute brain injury.