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Folia Neuropathologica
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2/2016
vol. 54
 
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Original paper

Ultrastructural pathology of human peritumoural oedematous cerebellar cortex

Orlando José Castejón

Folia Neuropathol 2016; 54 (2): 127-136
Online publish date: 2016/05/19
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Introduction

Some earlier research investigations have been reported on ultrastructural biology and pathology of cerebellar tumours [14,15,16,27]. Axonal torpedoes on Purkinje cells of the cerebellum have been observed at electron microscopic level in infantile neuroaxonal dystrophy, in two cases of brain tumours, and in a case of a 5-year-old boy suffering from juvenile astrocytoma [21,24,32]. The presence of intranuclear filamentous inclusions in cerebellar Golgi cells was reported by us in three patients with cerebellar tumours [4].
However, very few electron microscopic studies have been devoted to study the cortical biopsies of the cerebellar peritumoral region taken during neurosurgical treatment of human cerebellar, cerebellopontine and mesencephalic tumours for systematically examining the alterations of cerebellar neurons and their abnormal intracortical circuits. This study is basically important to understand the pathogenesis of clinical cerebellar syndromes, such as gait disturbance, tremor, nystagmus, compression of cranial nerves, cognitive and neurobehavioural changes observed in the affected patients.

Material and methods

The present study describes the ultrastructural changes of focal peritumoural cerebellar cortex by means of cortical biopsies immediately taken during neurosurgical treatment of seven patients with cerebellar, cerebellopontine and mesencephalic tumours. Table I contains the clinical data, neuropathological features and localization of tumours. The neurosurgical study was performed and the cortical biopsies were taken according to basic principles of Helsinki. Cerebellar cortical biopsies of seven patients, ranging from 2- to 50-years old, with clinical diagnosis of cerebellar haemangioma, mesencephalic meningioma, cerebellopontine astrocytoma, cerebellopontine meningioma, and medulloblastoma of cerebellar vermis were examined by means of conventional transmission electron microscopy. Two to five mm thick cortical biopsies were immediately fixed in the surgical room in 4% glutaraldehyde-0.1M phosphate or cacodylate buffer, pH 7.4, at 4°C. After 2 hours’ glutaraldehyde-fixation period, the cortical biopsies were divided into approximately 1 mm fragments and observed under a stereoscopic microscope to check the quality of fixation of the sample, the glutaraldehyde diffusion rate, and the brownish coloration of the surface and deeper cortical regions, indicative of good glutaraldehyde fixation by immersion technique. Immersion in fresh glutaraldehyde solution of 1 mm slices was secondarily done for 2 hours after eliminating the remaining blood from the cortical biopsy by washing in similar 0.1 M phosphate or cacodylate buffer, pH 7.4 to avoid oxidation of the primary fixative solution. Secondary fixation in 1% osmium tetroxide-0.1 M phosphate buffer, pH 7.4, was carried out for 1-2 hours at 4°C. Black staining of the cortical slices also was observed under a stereoscopic microscope to check osmium tetroxide diffusion rate and quality of secondary fixation. They were then rinsed for 5 to 10 minutes in phosphate or cacodylate buffer of similar composition to that used in the fixative solution, dehydrated in increasing concentrations of ethanol, and embedded in Araldite or Epon. For proper orientation during the electron microscope study and observation of cortical layers, approximately 0.1 to 1 µm thick sections were stained with toluidine blue and examined with a Zeiss photomicroscope. Ultrathin sections, obtained with Porter-Blum and LKB ultramicrotomes, were stained with uranyl acetate and lead citrate, and observed in a JEOL 100B transmission electron microscope (TEM) at magnifications ranging from 20,000x to 90,000x. For each cortical biopsy, approximately 50 electron micrographs were systematically studied. Digital images were Photoshop analysed [7]. This paper was carried out according to the ethical principles of Helsinki Declaration, Ethical Committee of Biological Research Institute. The relative written consent was obtained in each case.

Results

Close examination of cerebellar cortex showed at the level of granule cell layer, swollen granule cells characterized by an electron translucent cytoplasm and swollen mitochondria. There appeared closely apposed and their continuous limiting plasma membrane appeared separated by a 20 nm extracellular space (Fig. 1). The neighbouring mossy fibre glomerulus showed the oedematous afferent mossy fibre endings synapsing with swollen granule cell dendrites. Degenerated myelinated axons were observed at the granular layer. Some of these degenerated axons could correspond to the efferent Purkinje cell axons and the incoming afferent cerebellar mossy and climbing fibres (Fig. 2). Reactive astrocytes containing an increased amount of huge lipofuscin granules were found at the granular and molecular layers (Fig. 3).
The oedematous Golgi cell depicted an electron translucent cytoplasm, notably swollen mitochondria, dilated rough endoplasmic reticulum cisterns with focal detachment of associated ribosomes, enlarged flattened cisterns of Golgi apparatus, nu­merous lipofuscin granules, and intranuclear inclu­sions (Fig. 4).
At the level of Purkinje cell layer, clear and dark ischaemic Purkinje cell bodies exhibited a dilated smooth and rough endoplasmic reticulum with focal detachment of membrane associated ribosomes (Figs. 5 and 6). The Purkinje cell bodies appeared covered by the swollen Bergmann glial cell cytoplasm. Dense Purkinje cell dendrites can be traced throughout the width of molecular layer exhibiting the emergency sites of Purkinje dendritic spines, which establish axo-spino-dendritic synaptic contacts with the swollen synaptic varicosities of parallel fibres (Fig. 7). Oedematous Bergmann glial cells were observed containing swollen mitochondria and lipofuscin granules, and exhibiting the characteristic ascending Bergman fibres in the molecular layer (Fig. 8). The cytoplasm of Bergman glial cell also appeared surrounding the Purkinje recurrent axonal collaterals. In addition, swollen basket cells were found surrounding the Purkinje cell layer (Fig. 9).
The oedematous stellate neurons displayed swollen mitochondria, invaginated nuclear envelope, and enlarged rough endoplasmic reticulum with detachment of associated ribosomes (Fig. 10). Proteinaceous oedema fluid with fibrinous organization was found occupying the enlarged extracellular space of cerebellar molecular layer (Fig. 11).
Swollen longitudinal granule cell axons or parallel and climbing fibres were observed at the molecular layer alternating with the ascending and swollen Bergmann glial fibres, and clear dendritic processes of Golgi, basket and stellate cells. The parallel fibres were identified by the characteristic cross-sectioned bundles of parallel fibres and their degenerated “en passant” synaptic varicosities. In addition, the swollen cytoplasm of Bergman glial cell closely surrounds the degenerated parallel fibre-Purkinje dendritic spine synapses. The latter appeared as dark degenerated structures (Fig. 12). Swollen climbing fibre branches appeared as electron translucent unmyelinated axons ascending in the molecular layer and containing numerous microtubules and neurofilaments. The large climbing fibre synaptic ending showed a decreased number of synaptic vesicles closely aggregated toward the presynaptic membrane. They appeared making synaptic contacts with clear and oedematous processes, presumably corresponding to ascending dendrites of Golgi, stellate and basket cell dendrites. Swollen and reactive Bergmann glial cells were observed surrounding climbing fibre endings (Fig. 13).
Swollen and dark microglial cells were observed engulfing haematogenous serum proteinaceous oe­­dema fluid (Fig. 14). Some cerebellar capillaries showed a reduplicated basement membrane (Fig. 15), and open and apparently intact endothelial junctions. Similar findings also were reported by us in traumatic brain injuries [7].

Discussion

The present study describes the oedematous intracellular changes and degenerated intracortical circuits of cerebellar neurons, and the reactive changes and neuroglial cells of cerebellar cortex in different cerebellar and brain stem tumours examined. Swollen clear granule, Golgi, Purkinje, stellate and basket cells, and Bergmann glial were found. On the contrary, some dark ischaemic Purkinje cells were observed suggesting a major vulnerability of Purkinje cells to the tumour expansion forces and the sustained ischaemic process. Similar results in cerebellar neurons, mainly dark Purkinje cells and Bergmann glial cells as described above, were also reported earlier by Sobaniec-Lotowska [28] in experimental encephalopathy induced by chronic application of valproate. According to this author, the general pattern of submicroscopic alterations of Purkinje cell perikarya suggested severe disorders in several intercellular biochemical extents, including inhibition of oxidative phosphorylation and abnormal protein synthesis, both of which could lead to lethal damage. The enlargement of smooth and endoplasmic reticulum (ER) and the focal detachment of associated ribosomes suggest a form of stress-induced endoplasmic reticulum dysfunction [16]. According to Paschen [26], changes in neuronal calcium activity in the various subcellular compartments have divergent effects on affected cells. In the cytoplasm and mitochondria, where calcium activity is normally low, a prolonged excessive rise in free calcium levels is believed to be toxic. On the contrary, in the endoplasmic reticulum, calcium activity is relatively high and severe stress is caused by a depletion of ER calcium stores.
Dark Purkinje cell dendritic branches and dendritic spines were observed at the molecular layer. These findings herein interpreted as characterizing ischaemic Purkinje neurons, could also be due to activation of caspases and mitochondrial dysfunction [31], or hypoxia induced excitotoxic-type of dark cell degeneration [2]. Dark Purkinje dendritic arborisation and the degenerated parallel fibre and climbing Purkinje spines dendritic synapses could be related to the cognitive impairment and motor deficits of patients under study. Similar findings have been reported on dendritic Purkinje cells from human cerebellar vermis in Alzheimer’s disease [25].
The presence of intranuclear filamentous inclusions in cerebellar Golgi cells was earlier reported by Castejon and Arismendi [4] in three patients with cerebellar tumours. They were related with stress conditions, excitotoxicity, damage of mitochondrial respiratory chain and impaired energy metabolism [18].
Degenerated cerebellar afferent and efferent myelinated axons were found in the granular and molecular layers of cerebellar cortex, which provide evidence for myelin lipid dysfunction in the oedematous cerebellar cortex related with the oligodendrocyte degenerating hydropic changes [6,13]. Cytoskeletal disassembly and caspases are recently implicated in these processes highlighting the degenerating axonal processes in human hypoxic- ischaemic injury [29].
The presynaptic synaptic terminals of climbing fibres synapsing on Purkinje cell dendrites exhibited a decreased number of synaptic vesicles, indicating climbing fibre presynaptic degeneration [11]. Similar findings were reported by Baloyannis in vascular dementia [1].
We found lipofuscin-rich astrocyte which can be related with the associated ischaemic and degenerative processes. Similar observations have been reported by the present author in traumatic human brain injuries [9,10]. We also found reactive phagocytic astrocytes and microglial cells engulfing haematogenous proteinaceous oedema fluid. These findings have been also described by Stoll et al. [30] in ischaemic brain lesions, and by us in severe traumatic and complicated human brain injuries [5,9,10].

Clinical correlates of peritumoural ischaemic process

The electron microscopic findings described herein and related with the oedematous microneurons (granule, basket, and stellate cells) and macroneurons (Golgi and Purkinje cells) of cerebella cortex, and their degenerated synaptic contacts in both granule cell and molecular layers with cerebellar afferent and intrinsic fibres could be related with the tremor, nystagmus, dismetry, gait disturbance and neurobehavioral changes observed in the patients examined. According to Louise [21], a cascade of biochemical and cellular events occurring in the Purkinje cell layer and its neuron neighbourhood, as well as the physiological effects of secondary remodelling/rewiring could be related with essential tremor. The derangement of neurons and intracortical circuits is in agreement with the hypothesis that cerebellar damage impairs executive control and monitoring of movement generation [3].

Some molecular considerations on peritumoural cerebellar ischaemic process

The peritumoural ischaemic process seems to include the detrimental biochemical events postulat­ed by Hou and MacManus [17] for ischaemia-induced neuronal death, such as perturbation of calcium homeostasis leading to increased excitotoxicity, malfunction of endoplasmic reticulum and mitochondria, elevation of oxidative stress causing DNA damage, alteration in proapoptotic gene expression, and activation of the effector cysteine proteases (caspases) and endonucleases leading to the final degradation of the genome. The observed peritumoural cerebellar cell changes herein described also are similar to those described as permanent compression ischaemia [19]. A role of glutamate receptors in ischaemic process has been firmly established and a prominent characteristic of ischaemic insults is endoplasmic reticulum (ER) stress. In addition, a down-regulation of cell surface GABAB receptors has been postulated leading to diminished neuronal inhibition and contributing to excitotoxicity in cerebral ischaemia [23]. Cerebellar Purkinje neurons are selectively vulnerable to AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepriopionic acid)-induced delayed neurotoxicity known as dark cell degeneration that is expressed as cytoplasmic and nuclear condensation, neuron shrinkage, and failure of physiology [31]. Sustained activation of N-methyl-d-aspartate (NMDA)-type glutamate receptors leads to excitotoxic neuronal death as observed in stroke, brain trauma, and neurodegenerative disorders. Superoxide production by NADPH oxidase is a requisite event in the process leading from NMDA receptor activation to excitotoxic death [20].
Alterations of cerebellar excitatory and inhibitory synaptic transmission may contribute to the ischaemia-induced neuronal degeneration [22]. We have observed swollen mitochondria indicating a decrease in high energy phosphate produced by ischaemia, which causes depletion of the materials necessary to produce this phosphate and strongly affecting the electron transport chain. The biochemical pathology of swollen mitochondria in brain oedema have been earlier analysed in detail by us in a previous publication [12].
The presence of extracellular electron dense de­posits of proteinaceous oedema fluid and fibrinous organization, and the reduplicated capillary basement layering have also been described by us in severe traumatic brain injuries [7,8].

Acknowledgement

This paper has been carried out with a subvention obtained from CONDES-LUZ and Castejón Foundation. We deeply appreciate the technical digital help of Lic. Orlando Castejon de Pablos for preparation of electron micrographs.

Disclosure

Author reports no conflict of interest.

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Copyright: © 2016 Mossakowski Medical Research Centre Polish Academy of Sciences and the Polish Association of Neuropathologists. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0) License (http://creativecommons.org/licenses/by-nc-sa/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material, provided the original work is properly cited and states its license.
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